Novel Compounds for Imaging Tau Proteins That Accumulate in the Brain

ABSTRACT

The present invention provides a compound represented by the following formula (I), a pharmaceutically acceptable salt thereof, or a solvate thereof. 
     
       
         
         
             
             
         
       
     
     wherein:
         R 1  and R 2  are each separately selected from the group consisting of hydrogen, alkyl, alkenyl, acyl, and hydroxyalkyl;   R 3  is hydrogen or halogen;   ring A is a benzene ring or a pyridine ring;   ring B is selected from the group consisting of the following formulas (i), (ii), (iii), and (iv):       

     
       
         
         
             
             
         
       
         
         
           
             in the formula (ii), R a  is alkyl; 
             R 4  and R 5  are each separately selected from the group consisting of hydrogen, hydroxy, alkoxy, haloalkoxy, halohydroxyalkoxy, and aminoalkyl; and
 
[Formula 3]
 
 
 
represents a double bond or a triple bond. The above compound can be used as a molecular probe for imaging tau proteins that accumulate in the brain.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/346,914, filedMar. 24, 2014, which is a National Phase entry of PCT/JP2012/083286,filed Dec. 21, 2012, the disclosures of each of which are incorporatedby reference in their entireties.

TECHNICAL FIELD

The present invention relates to novel compounds for imaging tauproteins that accumulate in the brain, methods of preparing thecompounds, intermediates thereof, and methods of use thereof.

BACKGROUND

In many neurodegenerative diseases such as Alzheimer's disease (AD), tauprotein aggregates accumulate in brain cells, generally referred to as“tauopathies.” Of these, in familial frontotemporal lobar degeneration(FTLD) (known as frontotemporal dementia and Parkinsonism linked tochromosome 17 (FTDP-17)), genetic mutations in tau genes have beendiscovered. After that, a study of Tg mice that overexpressed human wildtype (WT) or FTDP-17 mutant tau proteins has made it clear that tauamyloid production takes part in the mechanism of neurodegenerativeepisodes in Alzheimer's disease (AD) and non-Alzheimer-type (non-AD)tauopathies (non-patent literature 1). Also, it has been shown that tauprotein aggregates in AD, referred to as neurofibrillary tangles (NFT),are closely linked to disease severity than senile plaques that are madeof amyloid β peptides (Aβ) (non-patent literature 2). By contrast withamyloid precursor protein (APP) Tg mice in which Aβ aggregatesaccumulate without a decrease of neurons, tau Tg mice exhibit asignificant decrease of neurons (non-patent literature 3). It istherefore necessary, in future studies, to make the neurotoxicity offibrous tau proteins in tauopathies pathologically clear, by acomparative evaluation of the living human brain and the mouse brain.

In vivo imaging—for example, positron emission tomography (PET), opticalimaging, and nuclear magnetic resonance imaging—is able to visualize Aβdeposits in AD patients and AD mouse models in vivo. As molecular probesto be used thereupon, compounds such as [¹⁸F]FDDNP,[¹¹C]6-OH-BTA-1(PIB), [¹¹C]AZD2184, [¹¹C]BF-227, [¹⁸F]-BAY94-9172, and[¹⁸F]AV-45 are known (patent literatures 1 to 4). Among these,[¹⁸F]FDDNP has been suggested to bind to both senile plaques and NFTs.However, since this compound has binding to the dense core of Aβaggregates, interactions with tau pathologies in AD patients have notbeen shown clearly. In addition, there is a problem that this compounddoes not bind to tau aggregates in non-AD tauopathy brains withoutsenile plaques, and therefore cannot directly show binding to taupathologies in vivo. Consequently, development of novel compounds thatspecifically bind to tau proteins that accumulate in the brain due to ADand non-AD tauopathies, and that allow imaging of tau aggregates, hasbeen sought after.

CITATION LIST Patent Literature

-   Patent Literature 1: Japanese Unexamined Patent Application    Publication (Translation of PCT Application) No. 2009-519239-   Patent literature 2: Japanese Unexamined Patent Application    Publication No. 2012-102106-   Patent literature 3: Japanese Unexamined Patent Application    Publication (Translation of PCT Application) No. 2011-516866-   Patent literature 4: Japanese Unexamined Patent Application    Publication (Translation of PCT Application) No. 2011-512354

Non-Patent Literature

-   Non-patent literature 1: Ballatore, C et al., Tau-mediated    neurodegeneration in Alzheimer's disease and related disorders, Nat.    Rev. Neurosci, 8, 663-72 (2007).-   Non-patent literature 2: Arriagada, P. V. et al., Neurofibrillary    tangles but not senile plaques parallel duration and severity of    Alzheimer's disease, Neurology 42, 631-639 (1992).-   Non-patent literature 3: Yoshiya, Y. et al., Synapse loss and    microglial activation precede tangles in a P301S tauopathy mouse    model., Neuron 53, 337-351 (2007).

SUMMARY OF INVENTION Technical Problem

It is an object of the present invention to provide novel compounds thatcan specifically bind to tau proteins that accumulate in the brain.

Solution to Problem

The present inventors have tested compounds of various dimensions forbinding to tau aggregates. As a result of this, it has been found outthat compounds having a basic structure of specific length ranging from13 to 19 Å exhibit affinity to tau aggregates in living organismsincluding AD and non-AD tauopathy patients. From this perspective, thepresent inventors have developed novel compounds that can specificallybind to tau aggregates.

The present invention provides a compound represented by the followingformula (I), a pharmaceutically acceptable salt thereof, or a solvatethereof:

wherein:

R₁ and R₂ are each separately selected from the group consisting ofhydrogen, alkyl, alkenyl, acyl, and hydroxyalkyl;

R₃ is hydrogen or halogen;

ring A is a benzene ring or a pyridine ring;

ring B is selected from the group consisting of the following formulas(i), (ii), (iii), and (iv):

in the formula (ii), Ra is alkyl;

R₄ and R₅ are each separately selected from the group consisting ofhydrogen, hydroxy, alkoxy, haloalkoxy, halohydroxyalkoxy, andaminoalkyl; and

[Formula 3]

represents a double bond or a triple bond. In one embodiment, in thecompound of the formula (I), one or more atoms are a radioisotope of theatom(s).

Advantageous Effects of Invention

The compounds of the present invention can specifically bind to tauaggregates. Consequently, it is possible to image tau proteins thataccumulate in the brain using the compounds of the present invention.

After being administered in mammals, the compounds of the presentinvention can quickly pass the blood brain barrier. The half-life of thecompounds of the present invention to last in the brain is approximately10 minutes, and therefore has an advantage of having little influence onthe human body. Also, the compounds of the present invention havefluorescence properties, so that the compounds of the present invention,when labeled with a radioactive isotope, are capable of double imaging,by the fluorescence properties and radioactivity of the compoundsthemselves.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B show confocal fluorescence images of frontal cortexslices of AD patients. FIG. 1A shows images that are stained with PIB,FSB and AT8, and with an anti-AβN3 (pE) antibody. FIG. 1B shows imagesthat are stained with PBB1 to PBB5, and with an anti-AβN3 (pE) antibody.

FIG. 2 shows double fluorescence staining images of AD NFTs and Pick'sdisease by FSB, PIB, THK523, FDDNP, BF-227, PBB1 to PBB5, and AT8.

FIG. 3A shows the results of in vitro and ex vivo labeling of NFT-liketau inclusions in PS19 mice using PBB1 to PBB5. FIG. 3B shows theresults of in vitro labeling of AD NFTs and NFT-like tau inclusions inPS19 mice using compounds other than PBB1 to PBB5.

FIGS. 4A-4J shows the results of non-invasive near-infrared imagingusing PBB5; FIG. 4A shows a reference autofluorescent signal (centerpanel) laid over a visible light image (left panel) of the shaved headpart of non-Tg WT mice. Elliptically-shaped regions of interest (ROIs)of the frontal cortex (FC), the brain stem (BS), and the cervical cord(SC) are shown in the right panel. FIG. 4B shows fluorescence intensitymaps of PBB5 (0.1 mg/kg) in 12-month-old WT mice (upper part) and PS19mice (lower part), before and 30 minutes and 240 minutes after theintravenous administration. FIGS. 4C, 4D and 4E show the ratios offluorescence intensity in the BS (c) and SC (d) ROIs, to the FC ROI, inthe WT mice (white: n=7) and the PS19 mice (black: n=7). FIG. 4F shows adistribution diagram of the ratios of SC and BS to FC 240 minutes later,against the number of FSB-positive NFT-like pathologies per unit area of20-μm tissue sections of the tau Tg mice. FIG. 4G shows the fluorescenceintensity (left) and the fluorescence duration (right) in 11-month-oldWT mice (upper part) and PS19 mice (lower part) 120 minutes after theintravenous injection of PPB5. FIG. 4H shows TPSF curve of SC and FCspots 120 minutes after injection in 11 month-old WT mice and Tg mice.FIG. 4I shows average durations of fluorescence in the FC, BS, and SCROIs in the WT mice (white; n=7) and Tg mice (black; n=7) 120 minutesafter the injection. FIG. 4J shows a distribution diagram of thefluorescence duration periods in the BS and SC ROIs 120 minutes afterthe injection, against the number of FSB-positive NFT-like pathologiesper unit area in 20 μm-thick tissue sections of the Tg mice.

FIGS. 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H, and 5I show real-time 2-photonlaser scanning images of PBB3 distribution at 0, 5, 20, 40, 80, 150, 300and 600 seconds after injection, respectively. FIGS. 5G and 5H show thatPBB3 remains bound to tau inclusions, while FIG. 5I shows that all PBB3had disappeared from WT mice within 300 seconds.

FIG. 6A shows the results of PET and autoradiographic detections of taupathologies of PS19 mice using [¹¹C]PBB2 and [¹¹C]PBB3; FIG. 6B showsautoradiograms of 20 μm-thick brain sections from non-Tg WT mice andPS19 mice treated with [¹¹C]PBB2 or [¹¹C]PBB3; FIG. 6C showssagittal-plane and coronal-plane PET images and MRI images, obtained byaveraging the dynamic scan data from 60 to 90 minutes after theintravenous administration of [¹¹C]PBB3. FIG. 6D shows FSB stain imagesof a brain section extracted from the PS19 mice after PET scanning (asagittal plane image (left panel) and a coronal plane image (centerpanel), and a high-magnification image (right panel)) of fibrous tauinclusions. FIG. 6E shows the time-activity curves (left panel) in thestriatums (ST) and brain stem (BS) of the PS19 mice and WT mice, and,the BS-to-ST ratios of radioactivity (right panel) (in each, n=5). FIGS.6H and 6I show ex vivo autoradiography images of the mice shown in FIGS.6F and 6G. FIGS. 6J and 6K show FSB stain images, using the same samplesas the samples from which the autoradiography images are obtained. FIG.6L shows time-activity curves in a plurality of brain tissues of WTmice. FIG. 6M shows the ratios of radioactivity in the brain stem to thestriatum, in PS19 mice (1 in the drawing) and WT mice (2 in the drawing)(n=5), over the imaging period.

FIG. 7 shows coronal-plane PET images in the brains of WT mice (leftpanel) and PS19 Tg mice (right panel) to which [¹¹C]mPBB5 is injected.

FIG. 8 shows autoradiography images (FIG. 8A) and PET images (FIG. 8B)of brain slices of AD patients using [¹¹C]PBB3 and [¹¹C]PIB.

DESCRIPTION OF EMBODIMENTS 1. Definitions

The term “alkyl” means a monovalent group that is produced whenaliphatic saturated hydrocarbon misses one hydrogen atom. An alkyl has,for example, 1 to 15 carbon atoms, and typically has 1 to 10, 1 to 8, 1to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 2 to 6 carbon atoms. An alkylmay be a straight chain or may be branched. Examples of alkyls include,but are by no means limited to, methyl, ethyl, propyl, isopropyl,2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl,3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl,2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl,2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl,2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl,isobutyl, t-butyl, pentyl, isopentyl, neopentyl, and hexyl. An alkyl mayfurthermore be substituted by an adequate substituent.

In this description, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1to 3, 1 to 2, 2 to 8, 2 to 6, 2 to 4, 3 to 8, 3 to 6, 4 to 8, and 4 to 6carbon atoms will be represented as C₁₋₁₅, C₁₋₁₀, C₁₋₈, C₁₋₆, C₁₋₅,C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₈, C₂₋₆, C₂₋₄, C₃₋₈, C₃₋₆, C₄₋₈, and C₄₋₆,respectively.

The term “cycloalkyl” means a monovalent group that is produced whenaliphatic saturated hydrocarbon forming a carbocyclic ring misses onehydrogen atom. A cycloalkyl has, for example, 3 to 10 carbon atoms, andtypically has 3 to 8, 3 to 6, 3 to 5, 3 to 4, 4 to 5, 4 to 6, or 4 to 8carbon atoms. Examples of cycloalkyls include, but are by no meanslimited to, cyclopropane, cyclobutane, cyclopentane, cyclohexane,cycloheptane, and cyclooctane. A cycloalkyl may furthermore besubstituted by an adequate substituent.

The term “alkenyl” means an unsaturated aliphatic hydrocarbon group thathas at least one double bond. An alkenyl has, for example, 2 to 15carbon atoms, and typically has, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to4, 2 to 3, 3 to 6, 3 to 8, 4 to 6, 4 to 7, or 4 to 8 carbon atoms. Analkenyl may be a straight chain or may be branched. Examples of alkenylsinclude, but are by no means limited to, to be specific, vinyl(—CH═CH₂), allyl (—CH₂CH═CH₂), —CH═CH(CH₃), —CH═C(CH₃)₂, —C(CH₃)═CH₂,—C(CH₃)═CH(CH₃), —C(CH₂CH₃)=CH₂, 1,3-butadienyl (—CH═CH—CH═CH₂), andhepta-1,6-diene-4-yl (—CH₂—(CH₂CH═CH₂)₂). An alkenyl may furthermore besubstituted by an adequate substituent.

The term “alkynyl” means an unsaturated aliphatic hydrocarbon group thathas at least one triple bond. An alkynyl has, for example, 2 to 15carbon atoms, and typically has 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4,2 to 3, 3 to 6, 4 to 6, 4 to 7, or 4 to 8 carbon atoms. An alkynyl maybe a straight chain or may be branched. Examples of alkynyls include,but are by no means limited to, ethynyl (—CECH), —CECH(CH₃),—CEC(CH₂CH₃), —CH₂CECH, —CH₂CEC(CH₃), and —CH₂CEC(CH₂CH₃). An alkynylmay furthermore be substituted by an adequate substituent.

The term “acyl” means a group that is represented by “—CO—R.” Here, Ris, for example, an alkyl, an alkenyl, or an alkynyl. Examples of acylsinclude, but are by no means limited to, acetyl (—COCH3), ethylcarbonyl,propylcarbonyl, pentylcarbonyl, cyclohexylcarbonyl, octylcarbonyl,2-ethylhexylcarbonyl, dodecylcarbonyl, phenylcarbonyl, benzylcarbonyl,naphthylcarbonyl and pyridylcarbonyl. An acyl may furthermore besubstituted by an adequate substituent.

The term “hydroxy” or “hydroxyl” means —OH. The term “hydroxyalkyl”means an alkyl group that is substituted by a hydroxy group (—OH).Examples of hydroxyalkyls include, but are by no means limited to,hydroxymethyl (—CH₂OH), 2-hydroxyethyl (—CH₂CH₂OH), 1-hydroxyethyl(—CH(OH)CH₃), 3-hydroxypropyl (—CH₂CH₂CH₂OH), 2-hydroxypropyl(—CH₂CH(OH)CH₃), and 1-hydroxypropyl (—CH(OH)CH₂CH₃). A hydroxyalkyl mayfurthermore be substituted by an adequate substituent. The term“halogen” or “halo” means fluoro (—F), chloro (—Cl), bromo (—Br), andiodine (—I).

The term “alkoxy” means an alkyl that is bound to other groups viaoxygen atoms (that is, —O-alkyl). Examples of alkoxys include, but areby no means limited to, methoxy (—O-methyl), ethoxy (—O-ethyl), propoxy(—O-propyl), —O-isopropyl, —O-2-methyl-1-propyl, —O-2-methyl-2-propyl,—O-2-methyl-I-butyl, —O-3-methyl-1-butyl, —O-2-methyl-3-butyl,—O-2,2-dimethyl-1-propyl, —O-2-methyl-1-pentyl, 3-O-methyl-1-pentyl,—O-4-methyl-1-pentyl, —O-2-methyl-2-pentyl, —O-3-methyl-2-pentyl,—O-4-methyl-2-pentyl, —O-2,2-dimethyl-1-butyl, —O-3,3-dimethyl-1-butyl,O-2-ethyl-1-butyl, —O-butyl, —O-isobutyl, —O-t-butyl, —O-pentyl, —O—isopentyl, O-neopentyl, and —O-hexyl. An alkoxy may furthermore besubstituted by an adequate substituent.

The term “haloalkyl” means an alkyl that is substituted by at least onehalogen. Haloalkyls include fluoroalkyl, chloroalkyl, bromoalkyl, andiodoalkyl. Examples of haloalkyls include, but are by no means limitedto, fluoromethyl, chloromethyl, bromomethyl, iodomethyl, fluoroethyl,chloroethyl, bromoethyl, iodoethyl, fluoropropyl, chloropropyl,bromopropyl, iodopropyl, fluorobutyl, chlorobutyl, bromobutyl,iodobutyl, fluoropentyl, chloropentyl, bromopentyl, iodopentyl,fluorohexyl, chlorohexyl, bromohexyl, iodohexyl, fluoroheptyl,chloroheptyl, bromoheptyl, iodoheptyl, fluorooctyl, chlorooctyl,bromooctyl, and iodooctyl. A haloalkyl may furthermore be substituted byan adequate substituent.

The term “haloalkoxy” means an alkoxy that is substituted by at leastone halogen (that is, —O-haloalkyl). Haloalkoxys include fluoroalkoxy,chloroalkoxy, bromoalkoxy, and iodoalkoxy.

The term “halohydroxyalkyl” means a hydroxyalkyl that is substituted byhalogen. Halohydroxyalkyls include fluorohydroxyalkyl,chlorohydroxyalkyl, bromohydroxyalkyl, and iodohydroxyalkyl. Examples ofhalohydroxyalkyls include 1-bromo-3-propanol, 1-iodo-3-propanol,1-bromo-2-ethanol, 1-iodo-2-ethanol, 1-bromo-1-methanol or1-iodo-1-methanol.

The term “halohydroxyalkoxy” means a haloalkoxy that is substituted by ahydroxy group. Halohydroxyalkoxys include fluorohydroxyalkoxy,chlorohydroxyalkoxy, bromohydroxyalkoxy, and iodohydroxyalkoxy. Examplesof halohydroxyalkoxys include —O—CH(F)(OH), —O—CH₂CH(F)(OH),—O—CH(OH—CH₂(F), —O—CH₂—CH(F)(OH), —O—CH(OH—CH₂—CH₂(F), —O—CH₂—CH(OH—CH₂(F), —O—CH₂—CH(OH)—CH₂ (F), —O—CH(CH₂—F)(CH₂OH) and—O—CH₂—CH₂—CH(F)(OH).

The term “nitro” means —NO₂. The term “amino” means —NH₂. The term“aminoalkyl” means an alkyl group that is substituted by an amino group.Examples of aminoalkyls include, but are by no means limited to,aminomethyl, aminoethyl, aminopropyl, aminoisopropyl, aminobutyl,aminopentyl, aminohexyl, and aminooctyl.

The term “substituent” means one or more atoms or an atomic group thatis introduced in a given chemical structural formula. Examples ofsubstituents include, for example, C₁₋₈ alkyls (methyl, ethyl, n-propyl,iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, or n-hexyl, orits isomer, and so on), C₂₋₈ alkenyls (vinyl, allyl, —CH═CH(CH₃),—CH═C(CH₃)₂, —C(CH₃)═CH₂, —C(CH₃)═CH(CH₃), —C(CH₂CH₃)═CH₂ and so on),C₂₋₈ alkynyl(ethynyl, —C═CH(CH₃), (CH₃), —CH₂C═C(CH₂CH₃), —CH₂CECH,—CH₂CEC(CH₃), —CH₂CEC(CH₂CH₃) and so on), alkoxy, hydroxy, halogen,haloalkyl, cycloalkyl (cyclopropyl, cyclobutyl, cyclopentyl, orcyclohexyl and so on), amino, nitro, acyl (acetyl and so on) (—COCH₃),carboxyl (—COOH), ester (—COOR^(x), where Rx is C₁₋₆ alkyl and so on),amide (—CONR^(y)R^(z), where R^(y) and R^(z) are individually H or C₁₋₆alkyl and so on), thiol (—SH), sulfonic acid (—SO₃H), nitrile (—CN),aromatic rings (aryl, phenyl, benzoyl, or naphthalenyl and so on),heterocyclic rings (pyrrolidinyl, tetrahydrofuranyl, pyrrolyl, furanyl,thiophenyl, piperidinyl, oxanyl, or pyridinyl and so on), and so on.

The term “pharmaceutically acceptable salt” means a salt that is notharmful to mammals, especially humans. Pharmaceutically acceptable saltscan be formed using non-toxic acids or bases, including mineral acids orinorganic bases, or organic acids or organic bases. Examples ofpharmaceutically acceptable salts include metal salts formed withaluminum, calcium, lithium, magnesium, potassium, sodium, zinc and soon, and organic salts formed with lysine, N,N′-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine(N-methylglucamine), procaine and so on. Also, pharmaceuticallyacceptable salts contain acid-addition salts and base-addition salts.

The term “pharmaceutically acceptable carriers” means pharmaceuticallyacceptable materials, compositions, or vehicles such as physiologicalsaline solutions, liquid or solid fillers, diluents, solvents, orencapsulants. Examples of pharmaceutically acceptable carriers includewater, saline water, physiological saline water or phosphate bufferedsaline water (PBS), sodium chloride injection solution, Ringer'sinjection solution, isotonic dextrose injection solution, sterile waterinjection solution, dextrose, and lactated Ringer's injection solution.

The term “effective dose” refers to the amount of a compound or acomposition which will have a targeted effect. For example, in someembodiments, the effective dose may refer to the amount of a compound ora composition which will enable tau imaging.

The term “solvate” means a solvent-containing compound that is formed byassociation of one or a plurality of solvent molecules to the compoundsof the present invention. Solvates include, for example, monosolvates,disolvates, trisolvates, and tetrasolvates. Also, solvates includehydrates. The term “hydrate” means a compound further containing astoichiometric or a non-stoichiometric amount of water constrained bynon-covalent bonding intermolecular force, or a salt thereof. Hydratesinclude monohydrates, dihydrates, trihydrates, and tetrahydrates.

The term “treatment” means moderating or remitting the progress,severity and/or period of a disease or condition. The term “prevention”means reducing the danger of catching or making worse a predetermineddisease or condition, or reducing or suppressing the recurrence, startor progress of a predetermined disease or condition, or one or aplurality of symptoms.

The term “tau imaging” means imaging tau proteins that accumulate in thebrain. This imaging may be performed by positron emission tomography(PET), fluorescence microscopy measurement, multi-photon imaging,two-photon imaging, near-infrared fluorescence imaging, autoradiography,and single-photon emission computed tomography (SPECT).

2. Compounds of the Present Invention

The present invention provides a compound represented by the followingformula (I), a pharmaceutically acceptable salt thereof, or a solvatethereof:

Wherein:

R₁ and R₂ are each separately selected from the group consisting ofhydrogen, alkyl, alkenyl, acyl, and hydroxalkyl;

R₃ is hydrogen or halogen;

ring A is a benzone ring or a pyridine ring;

ring B is selected from the group consisting of the following formulas(i), (ii), (iii), and (iv):

in the formula (ii), R_(a) is alkyl;

R₄ and R₅ are each separately selected from the group consisting ofhydrogen, hydroxyl, alkoxy, haloalkoxy, halohydroxyalkoxy, andaminoalkyl; and

[Formula 6]

represents a double bound or a triple bond.

In one embodiment, ring B is the formula (i) or the formula (ii). Inanother embodiment, ring B is the formula (i). In yet anotherembodiment, ring B is the formula (ii). When ring B is the formula (ii),the type of the counter anion is not particularly limited, and may bep-toluenesulfonate, I⁻ and or the like. In one embodiment, ring B is theformula (iii). In another embodiment, ring B is the formula (iv).

When ring B is the formula (1), R₄ and R₅ can be at substitutablepositions in the benzothiazole ring of the formula (1). Preferably, R₄and R₅ are at position 6 and position 5 in the benzothiazole ring of theformula (i), respectively. When ring B is the formula (ii), R₄ and R₅can be at substitutable positions in the benzothiazolium ring of theformula (ii). Preferably, R₄ and R₅ are at position 6 and position 5 inthe benzothiazolium ring of the formula (ii), respectively. When ring Bis the formula (iii), R₄ and R₅ can be at substitutable positions in thebenzofuran ring of the formula (iii). Preferably, R₄ and R₅ are atposition 5 and position 6 in the benzofuran ring of the formula (iii),respectively. When ring B is the formula (iv), R₄ and R₅ can be atsubstitutable positions in the quinoline ring of the formula (iv).Preferably, R₄ and R₅ are at position 6 and position 7 in the quinolinering of the formula (iv), respectively.

In one embodiment, ring A is a pyridine ring. In another embodiment,ring A is a benzene ring. Preferably, ring A is the pyridine ringrepresented by the following structural formula, in the orientation ofthe structural formula of the formula (I).

In one embodiment, R₁ and R₂ are both hydrogen. In one embodiment, R₁and R₂ are each separately hydrogen or alkyl, especially C₁₋₈ alkyl, andpreferably methyl. In another embodiment, R₁ is hydrogen, R₂ is alkyl,especially C₁₋₆ alkyl, and preferably methyl. In yet another embodiment,R₁ and R₂ are both alkyl, especially C₁₋₆alkyl, and preferably methyl.

In one embodiment, R₁ and R₂ are each separately hydrogen or alkenyl,especially C₂₋₈ alkenyl, and preferably allyl (—CH₂CH═CH₂) orhepta-1,6-diene-4-yl (—CH₂—(CH₂CH═CH₂)₂). In another embodiment, R₁ ishydrogen, R₂ is alkenyl, especially C₁₋₈ alkenyl, and preferably allyl(CH₂CH═CH₂) or hepta-1,6-diene-4-yl (—CH₂—(CH₂CH═CH₂)₂). In yet anotherembodiment, R₁ and R₂ are both alkenyl, especially C₁₋₈ alkenyl, andpreferably allyl (—CH₂CH═CH₂) or hepta-1,6-diene-4-yl(—CH₂—(CH₂CH═CH₂)₂).

In one embodiment, R₁ and R₂ are each separately hydrogen or acyl,especially C₁₋₈ acyl, and preferably acetyl (—COCH₃). In anotherembodiment, R₁ is hydrogen, R₂ is acyl, especially C₁₋₈ acyl, andpreferably acetyl (—COCH₃). In yet another embodiment, R₁ and R₂ areboth acyl, especially C₁₋₈ acyl, and preferably acetyl (—COCH₃).

In one embodiment, R₁ and R₂ are each separately hydrogen orhydroxyalkyl, especially hydroxyC₁₋₈alkyl, preferably hydroxypropyl, andmore preferably 3-hydroxypropyl (—CH₂CH₂CH₂OH). In another embodiment,R₁ is hydrogen, R₂ is hydroxyalkyl, especially hydroxyC₁₋₈alkyl,preferably hydroxypropyl, and more preferably 3-hydroxypropyl(—CH₂CH₂CH₂OH). In yet another embodiment, R₁ and R₂ are bothhydroxyalkyl, especially hydroxyCi-salkyl, preferably hydroxypropyl, andmore preferably 3-hydroxypropyl (—CH₂CH₂CH₂OH).

In one embodiment, R₃ is hydrogen. In another embodiment, R₃ is halogen,that is, F, Cl, Br or I. Preferably, R₃ is F. Preferably, R₃ is ¹⁸F. Inthe orientation of the structural formula of the formula (I), R₃preferably assumes the following position.

In the orientation of the structural formula of the formula (I), ring Aand R₃ preferably have the following relationship.

In one embodiment, R_(a) is alkyl, preferably C₁₋₈ alkyl, and morepreferably methyl or ethyl. In one embodiment, R₄ and R₅ are bothhydrogen. In one embodiment, R₄ and R₅ are each separately hydrogen orhydroxy. In another embodiment, R₄ is hydroxy, and R₅ is hydrogen. Inyet another embodiment, R₄ is hydrogen, and R₅ is hydroxy. In yetanother embodiment, R₄ and R₅ are both hydroxy.

In one embodiment, R₄ and R₅ are each separately hydrogen or alkoxy,especially methoxy. In another embodiment, R₄ is alkoxy, especiallymethoxy, and R₅ is hydrogen. In yet another embodiment, R₄ is hydrogen,R₅ is alkoxy, especially methoxy. In yet another embodiment, R₄ and R₅are both alkoxy, especially methoxy.

In one embodiment, R₄ and R₅ are each separately hydrogen orhalohydroxyalkoxy, especially fluorohydroxyalkoxy, preferablyfluorohydroxyC₁₋₃alkoxy, and more preferably —O—CH₂—CH(OH)—CH₂(F) or—O—CH(CH₂—F)(CH₂OH). In another embodiment, R₄ is hydrogen, R₅ ishalohydroxyalkoxy, especially fluorohydroxyalkoxy, preferablyfluorohydroxyC_(l-3)alkoxy, more preferably —O—CH₂—CH(OH)—CH₂(F) or—O—CH(CH₂—F)(CH₂OH). In yet another embodiment, R₄ and R₅ are bothhalohydroxyalkoxy, especially fluorohydroxyalkoxy, preferablyfluorohydroxyC₁₋₃alkoxy, more preferably —O—CH₂—CH(OH)—CH₂(F) or—O—CH(CH₂—F)(CH₂OH). In yet another embodiment, R₄ and R₅ are bothhalohydroxyalkoxy, especially fluorohydroxyalkoxy, preferablyfluorohydroxyC₁₋₃alkoxy, and more preferably —O—CH₂—CH(OH)—CH₂(F) or—O—CH(CH₂—O(CH₂OH). In one embodiment, fluorohydroxyalkoxy contains aradioisotope. Preferably, this fluorohydroxyalkoxy is—O—CH₂—CH(OH)—CH₂(¹⁸F) or —O—CH(CH₂ ⁻⁸F)(CH₂OH).

In one embodiment, R₄ and R₅ are each separately hydrogen or aminoalkyl,especially aminomethyl or aminoethyl. In another embodiment, R₄ isaminoalkyl, especially aminomethyl or aminoethyl, and R₅ is hydrogen. Inyet another embodiment, R₄ is hydrogen, and R₅ is aminoalkyl, especiallyaminomethyl or aminoethyl. In yet another embodiment, R₄ and R₅ are bothaminoalkyl, especially aminomethyl or aminoethyl.

In one embodiment,

[Formula 10]

is a double bond. In another embodiment,

[Formula 11]

is a triple bond.

In one embodiment, the following compound is excluded from the compoundof the formula (I).

In one embodiment, the compound of the formula (I) is a compoundrepresented by the following formula (II):

wherein R₁ to R₅, and

[Formula 14]

have been defined above in the compound of the formula (I).

In one embodiment, the compound of the formula (I) is a compoundrepresented by the following formula (III):

wherein R₁ to R₅, and

[Formula 16]

have been defined in the compound of the formula (I).

In one embodiment, the compound of the formula (I) is a compoundrepresented by the following formula (IV).

wherein R₁ to R₅, R_(a) and

[Formula 18]

have been defined in the compound of the formula (I).

In one embodiment, the compound of the formula (I) is a compoundrepresented by the following formula (V).

wherein R₁ to R₅, and

[Formula 20]

have been defined in the compound of the formula (I).

In one embodiment, the compound of the formula (I) is a compoundrepresented by the following formula (VI).

wherein R₁, to R₅, and

[Formula 22]

have been defined in the compound of the formula (I).

In one embodiment, in the compounds of the formulas (I) to (VI), one ormore atoms are a radioisotope of the atom(s). The radioisotope may beselected from the group consisting of ¹⁵O, ¹³N, ¹¹C, ¹⁸F and so on, butis not particularly limited. Preferably, the radioisotope is ¹¹C or ¹⁹F.Of these, considering that the half-life of ¹¹C is approximately 20minutes and the half-life of ¹⁸F is approximately 110 minutes, acompound that is labeled with ¹⁸F may have a higher commercial value.Consequently, most preferably, the radioisotope is ¹⁸F.

Preferably, one or more of R₁ to R₅ are groups that contain aradioisotope. More preferably, one or both of R₁ and R₂ are groups thatcontain a radioisotope, and are, for example, groups that contain ¹¹C(for example, [¹¹C]alkyl to contain ¹¹CH₃). Even more preferably, R₃ isa group to contain a radioisotope, and is, for example, ⁻¹⁸F. Morepreferably, one or both of R₄ and R₅ are groups that contain aradioisotope, and are, for example, groups to contain ¹¹C (for example,[¹¹C]alkoxy to contain —O—CH₂—CH(OH)—CH₂(¹⁸F) and —O—CH(CH₂⁻¹⁸F(CH₂OH)). Here, [¹¹C]alkyl indicates that one or more carbon atomsin the carbon atoms constituting alkyl are ¹¹C. [¹¹C]alkoxy indicatesthat one or more carbon atoms in the carbon atoms constituting alkoxyare ¹¹C. [¹⁸F]fluorohydroxyalkoxy means a group in which 18F is bound tohydroxyalkoxy.

Specific examples of the compounds of the present invention include thefollowing compounds:

TABLE 1 Syn-

thesis property Embod- binding Name Name of Compound Structural Formulaiment capacity (1) PBB1 4-((1E,3E)-4- (benzo[d]thiazole-2-yl)buta-1,3-dienyl)-N,N- dimethylaniline

 1 (2) PBB2 2-((1E,3E)-4-(4-(methylamino) phenyl)buta-1,3-dienyl)benz[d]thiazole-6-ol

 2 (3) PBB3 2-((1E,3E)-4-(6-(methylamino) pyridine-3-yl)buta-1,3-dienyl)benz[d]thiazole-6-ol

 3 (4) PBB4 2-((1E,3E)-4-(6-(methylamino) pyridine-3-yl)buta-1,3-dienyl)benz[d]thiazole-5,6-diol

 4 (5) PBB5 2-((1E,3E)-4- (4-(dimethylamino) phenyl)buta-1,3-dienyl)-3-ethylbenzo[d]thiazole-3- ium

— (6) mPBB5 2-((1E,3E)-4- (4-(dimethylamino) phenyl)buta-1,3-dienyl)-3-ethyl-6-methoxybenzo[d] thiazole-3-ium

 5 (7) PBB2.1 (E)-2-(4-(4-(dimethylamino) phenyl)buta-1-en-3-ynyl)benz[d]thiazole-6-ol

 6 (8) PBB2.2 (E)-2-(4-(4-(methylamino) phenyl)buta-1-en-3-ynyl)benz[d]thiazole-6-ol

 7 (9) PBB2.3 (E)-2-(4-(4-(aminophenyl)buta-1-en-3-ynyl)benz[d]thiazole- 6-ol

 8 (10) PBB3.1 (E)-2-(4-(6-(dimethylamino) pyridine-3-yl)buta-1-en-3-ynyl)benz[d]thiazole-6-ol

 9 (11) PBB3.2 (E)-2-(4-(6-(methylamino) pyridine-3-yl)buta-1-en-3-ynyl)benz[d]thiazole-6-ol

10 (12) PBB3.2N (E)-5-(4-(6-(aminomethyl)benz[d]thiazole-2-yl)buta-3-en-1- ynyl)-N-methylpyridine-2- amine

11 (13) Core1-4 2-((1E,3E)-4-(4-aminophenyl) buta-1,3-dienyl)-6-methoxybenzo[d]thiazole- 5-ol

12 (14) Core1-5 N-(4-((1E,3E)-4-(5,6- dimethoxybenzo[d]thiazole-2-yl)buta-1,3-dienyl)phenyl) acetamide

13 (15) Core1-11 3-(4-((1E,3E)-4-(5,6- dimethoxybenzo[d]thiazole-2-yl)buta-1,3-dienyl) phenylamino)propan-1-ol

14 (16) Core1-15 4-((1E,3E)-4-(5,6- dimethoxybenzo[d]thiazole-2-yl)buta-1,3-dienyl)-N- isopropylaniline

15 (17) Core1-20 4-((1E,3E)-4-(5,6- dimethoxybenzo[d]thiazole-2-yl)buta-1,3-dienyl)-N- (hepta-1,6-diene-4-yl) aniline

16 (18) Core2-9 N-(5-((1E,3E)-4-(5,6- dimethoxybenzo[d]thiazole-2-yl)buta-1,3-dienyl)pyridine- 2-yl)acetamide

17 (19) Core2-10 3-(5-((1E,3E)-4-(5,6- dimethoxybenzo[d]thiazole-2-yl)buta-1,3-dienyl)pyridine- 2-ylamino)propan-1-ol

18 (20) Core2-14 N,N-diallyl-5-((1E,3E)-4-(5,6-dimethoxybenzo[d]thiazole- 2-yl)buta-1,3-dienyl)pyridine- 2-amine

19 F0-PBB3 analog 1-fluoro-2-(2-((1E,3E)-4-(6-(dimethylamino)pyridine-3- yl)buta-1,3-dienyl)benz[d]thiazole-6-yloxy)-2- hydroxymethyl-ethane

20-1 (21) F0-PBB3 1-fluoro-3-(2-((1E,3E)-4-(6-(methylamino)pyridine-3-yl) buta-1,3-dienyl) benz[d]thiazole-6-yloxy)propan-2-ol

20-2 (22) F0-PBB3.2 (E)-1-fluoro-3-(2-(4-(6- (methylamino)pyridine-3-yl)buta-1-en-3-ynyl)benz[d] thiazole-6-yloxy)propan-2-ol

21 (23) F1-PBB3 2-((1E,3E)-4-(2-(fluoro-6- (methylamino)pyridine-3-yl)buta-1,3-dienyl) benz[d]thiazole-6-ol

22 (24) F1-PBB3.2 (E)-2-(4-(2-fluoro-6- (methylamino)pyridine-3-yl)-buta-1-en-3-ynyl)benz[d] thiazole-6-ol

23 (25) F1-PBB

3 2-((1E,3E)-4-(2-fluoro-6- (methylamino)pyridine-3-yl)buta-1,3-dienyl)benzofuran- 5-ol

24 (26) F1-PB

.2 (E)-2-(4-(2-fluoro-6- (methylamino)pyridine-3-yl)-buta-1-en-3-ynyl)benzfuran- 5-ol

25 (27) PBQ1.0 2-((1E,3E)-4- (6-(dimethylamino) pyridine-3-yl)buta-1,3-dienyl)quinoline-6-ol

26 (28) PBQ3 2-((1E,3E)-4-(6-methylamino) pyridine-3-yl)buta-1,3-dienyl)quinoline-6-ol

27 (29) PBQ3.1 (E)-2-(4-(6-(dimethylamino) pyridine-3-yl)buta-1-en-3-ynyl)quinoline-6-ol

28 (30) PBQ3.2 (E)-2-(4-(6-(methylamino) pyridine-3-yl)buta-1-en-3-ynyl)quinoline-6-ol

29

indicates data missing or illegible when filed

In one embodiment, in the specific compounds given above, one or moreatoms are a radioisotope of the atom(s). Preferably, a carbon atom onnitrogen bound to a benzene ring or a pyridine ring is the radioisotope¹¹C. Preferably, F in the above specific compounds is the radioisotope¹⁸F. Preferably, a carbon atom of a methoxy group bound to abenzothiazole ring is the radioisotope ¹¹C. More preferably, an atomwith the “*” symbol in the structural formulas of the above specificcompounds (where there are two “*” symbols in a structural formula, oneor two of them) is the radioisotope of that atom, which is, for example,¹¹C or ¹⁸F. In this description, names such as [¹¹C]PBB3 mean that ¹¹Cis above the atom of the “*” symbol in the structural formula of PBB3,and so on.

3. Methods of Preparing the Compounds of the Present Invention SynthesisExample 1

The compound of the present invention according to the formula (I) canbe prepared according to following scheme 1:

In the above formulas, A, B, R₁ to R₅, and

[Formula 24]

have been defined about in the compound of the formula (I), and Hal ishalogen, especially bromo.

The method of preparing the compounds of the present invention includesstep 2 of reacting the compound (c1) with NHR₁R₂ and obtaining thecompound of the formula (I). Preferably, the method of preparing thecompounds of the present invention includes step 1 of coupling thecompound (a1) with the compound (b1) and obtaining the compound (c1),and step 2 of reacting the compound (c1) with NHR₁R₂ and obtaining thecompound of the formula (I).

The reaction of above step 1 can be performed under Wittig reactionconditions. This reaction can be performed under an inert gas atmospheresuch as argon or nitrogen. This reaction preferably uses bases such assodium hydride, sodium methoxide, or sodium ethoxide. This reaction ispreferably performed in an inert solvent such as tetrahydrofuran (THF)or N,N-dimethylformamide (DMF). The temperature of this reaction is notlimited, but can be in a range from 0° C. (in an ice bath) to roomtemperature.

The reaction of above step 2 can be performed under electrophilicaromatic substitution conditions. This reaction can be performed usingbases such as triethylamine. This reaction is preferably performed in aninert solvent such as DMF, or in an alcohol solvent such as methanol orethanol. The temperature of this reaction is not limited and ranges from0° C. (in an ice bath) to reflux temperature, and can be, for example,0° C. to 160° C., 30° C. to 150° C., 60° C. to 140° C., 90° C. to 130°C., or 120° C.

If necessary, it is possible to protect each compound with a protectinggroup prior to the reactions of above step 1 and/or step 2, and thenperform the reactions. When one or more of R₁ to R₅ have a hydroxy or anamino group, it is preferable to protect this hydroxy or amino groupwith an adequate protecting group. Examples of protecting groups forhydroxy or amino groups include alkyl groups such as methyl groups andethyl groups, benzyl groups, t-butyldimethylsilyl groups(—Si(CH₃)₂(t-C₄H₉)), tert-butoxycarbonyl groups (Boc: —COO-(t-C₄H₉)),methoxymethyl groups (—CH₂OCH₃), and ethoxymethyl groups (—CH₂OCH₂CH₃).Deprotection may be performed in adequate steps, in a method that isknown to one of skill in the art.

Synthesis Example 2

The compound of the present invention according to the formula (I), inwhich R₁ and R₂ are hydrogen, may be prepared in accordance withfollowing scheme 2:

In the above formulas, A, B, R₃ to R₅, and

[Formula 26]

have been defined above in the compound of the formula (I).

The method of preparing the compounds of the present invention includesstep 2 of reducing the compound (c2) and obtaining the compound of theformula (I-i) (the compound of the formula (I), in which R₁ and R₂═H).Also, prior to step 2, the method of preparing the compounds of thepresent invention further includes step 1 of coupling the compound (a2)with the compound (b2) and obtaining the compound (c2).

The reaction of above step 1 can be performed under Wittig reactionconditions. This reaction can be performed under an inert gas atmospheresuch as argon or nitrogen. This reaction preferably uses bases such assodium hydride, sodium methoxide, or sodium ethoxide. This reaction ispreferably performed in an inert solvent such as tetrahydrofuran (THF)or N,N-dimethylformamide (DMF). The temperature of this reaction is notlimited and might range from O° C. (in an ice bath) to room temperature.

The reduction in above step 2 can be performed under reducing conditionsto convert an aromatic nitro group into an amino group. For example,this reduction can be performed using iron, zinc, or tin chloride in anacid solution. For the acid solution, acetic acid, hydrochloric acid, ora liquid mixture of these may be used. Furthermore, salts such asammonium chloride may be used. This reduction c can be performed in analcohol solution such as methanol, ethanol or propanol. This reductioncan be performed in, but is by no means limited to, room temperature toreflux temperature. For example, this reduction can be performed at 20°C. to 100° C., 40° C. to 90° C., or 80° C. Also, this reduction can beperformed in catalytic hydrogenation using a metal catalyst such asplatinum, or can be performed in reduction using a metal hydride such aslithium aluminum hydride.

If necessary, it is possible to protect each compound with an adequateprotecting group prior to the reactions of above step 1 and/or step 2,and then perform the reactions. When one or more of R₃ to R₅ have ahydroxy or an amino group, it is preferable to protect that hydroxy oramino group with an adequate protecting group. Examples of protectinggroups for hydroxy or amino groups include alkyl groups such as methylgroups or ethyl groups, benzyl groups, t-butyldimethylsilyl groups(—Si(CH₃)₂(t-C₄H₉)), tert-butoxycarbonyl groups (Boc: —COO-(t-C₄H₉)),methoxymethyl groups (—CH₂OCH₂CH₃), and ethoxymethyl groups(—CH₂OCH₂CH₃). Deprotection may be performed in adequate steps, in amethod that is known to one of skill in the art.

Synthesis Example 3

The compound of the present invention according to the formula (I), inwhich R₁ is not hydrogen and R₂ is hydrogen, can be prepared accordingto following scheme 3:

In the above formulas, A, B, R₁, R₃ to R₅, and

[Formula 28]

have been defined above in the compound of the formula (I), where,however, R₁ is not hydrogen, X is an elimination group and is, forexample, halogen such as Cl, Br or I, alkoxy such as methoxy or ethoxy,triflate (—OSO₂—CF₃), carboxylate (—OCO—R), or an azide group (—N₃).

In scheme 3, the compound of the formula (I-i) which is the startingsubstance, can be synthesized according to above scheme 2. With themethod of preparing the compounds of the present invention, it ispossible to include step 1 of obtaining the compound of the formula (thecompound of the formula (I), in which R₁≠H and R₂═H) by reacting thecompound of the formula (I-i) with R₁X with reference to above scheme 3.

The reaction of above step 1 is alkylation, alkenylation, acylation orhydroxyalkylation of an amino group. When R₁ is alkyl, alkenyl,hydroxyalkyl and/or the like, this reaction can be performed undernucleophilic substitution reaction conditions. In this case, X ispreferably halogen, especially Cl, Br or I, or triflate (—OSO₂—CF₃).This reaction may use a base such as K₂CO₃ or triethylamine, or may usea reducing agent such as sodium hydride or sodium borohydride. Thisreaction may be performed under an inert atmosphere such as nitrogen orargon. This reaction may be performed in an inert solvent such asdichloromethane, chloroform, or N,N-dimethylformamide, or in an alcoholsolvent such as methanol or ethanol. This reaction can be performed at,but is not limited to, 0° C. (in an ice bath) to room temperature, or atroom temperature to reflux temperature, which can be, for example, 0° C.to 160° C., 30° C. to 150° C., 60° C. to 140° C., 90° C. to 130° C., or120° C.

In the reaction of above step 1, when R₁ is methyl in the formula(I-ii), a different method, in which the compound of the formula (I-i)is reacted with formaldehyde or paraformaldehyde, and, after that, theproduct is reduced using a reducing agent such as sodium hydride orsodium borohydride, may be used.

The reaction of above step 1 can be performed under nucleophilic acylsubstitution reaction conditions when R₁ is acyl and/or the like. Inthis case, X is preferably halogen such as Cl, Br or I, alkoxy such asmethoxy or ethoxy, carboxylate (—OCO—R), or an azide group (—N₃). Thisreaction can be performed in the presence of bases such as K₂CO₃ ortriethylamine. This reaction may be performed under acid conditions suchas HCl. This reaction can be performed in an inert solvent such asdichloromethane, chloroform or N,N-dimethylformamide. This reaction canbe performed at, but is by no means limited to, 0° C. (in an ice bath)to reflux temperature.

If necessary, it is possible to protect each compound with an adequateprotecting group prior to the reaction of above step 1, and then performthe reaction. When one or more of R₁, R₃ to R₅ have a hydroxy or anamino group, it is preferable to protect that hydroxy or amino groupwith an adequate protecting group. Examples of protecting groups forhydroxy or amino groups include alkyl groups such as methyl groups andethyl groups, benzyl groups, t-butyldimethylsilyl groups(—Si(CH₃)₂(t-C₄H₉)), tert-butoxycarbonyl groups (Boc: —COO-(t-C₄H₉)),methoxymethyl groups (—CH₂OCH₃), and ethoxymethyl groups (—CH₂OCH₂CH₃).Deprotection may be performed in a method that is known to one of skillin the art.

For example, when one or both of R₄ and R₅ are OH, as shown in steps 2to 4 of scheme 3, it is possible to synthesize protector 1 of theformula (I-i) from the compound of the formula (I-i), react it with R₁X,and, after that, synthesize the compound of the formula (I-ii) bydeprotection. Step 2 shown in scheme 3 is a step for when R₄ alone isOH. One of skill in the art should readily understand that the protectorcan be synthesized when R₅ alone is OH or when R₄ and R₅ are both OH.This protector can be obtained by reacting the compound of the formula(I-i) with t-butyldimethylchlorosilane. This reaction may use a basesuch as imidazole. This reaction is preferably performed under an inertgas atmosphere such as nitrogen or argon. Also, this reaction is usuallyperformed under an inert solvent such as dimethylsulfoxide. Thetemperature of this reaction is preferably room temperature.

After formula (I-i) protector 1 is prepared, a formula (I-ii) protectorcan be prepared by a reaction with R₁X (step 3). This reaction may adoptthe same reaction conditions as in step 1 above. After that, bydeprotecting the formula (I-ii) protector, the compound of the formula(I-ii), in which one or both of R₄ and R₅ are OH, can be obtained. Thisdeprotection can be performed using acid such as hydrochloric acid orusing fluoride ion such as tetra-n-butylammonium fluoride hydrate.

Also, for example, when one or both of R₄ and R₅ are aminoalkyl, asshown in steps 5 to 7 of scheme 3, it is possible to synthesize formula(I-i) protector 2 from the compound of the formula (I-i), react it withR₁X, and, after that, synthesize the compound of the formula (I-ii) bydeprotection. Step 5 shown in scheme 3 is a step for when R₄ alone isaminoalkyl. One of skill in the art should readily understand that theprotector can be synthesized when R₅ alone is aminoalkyl, as well aswhen R₄ and R₅ are both aminoalkyl. This protector can be obtained byreacting the compound of the formula (I-i) with tert-butyldicarbonate.

In scheme 3, when R₁X is [¹¹C]alkyl-X such as ¹¹CH₃—X, [¹¹C]alkly suchas ⁻¹¹CH₃ can be introduced.

Synthesis Example 4

The compound of the present invention according to the formula (I) inwhich R₁ is not hydrogen and R₂ is hydrogen, may be prepared accordingto following scheme 4.

In the above formulas, A, B, R₁, R₃ to R₅, and

[Formula 30]

have been defined above in the compound of the formula (I), in which,however, R₁ is not hydrogen.

The method of preparing the compounds of the present invention includesstep 2 of reacting the compound (c4) with Lewis acid and obtaining thecompound of the formula (I-ii) (the compound of the formula (I), inwhich R₁≠H and R₂═H). Furthermore, step 3 of obtaining the compound ofthe formula (I-iii) (the compound of the formula (I), in which R₁ andR₂≠H) by a reaction with R₂X after reduction, may be included. Also, themethod of preparing the compounds of the present invention may furtherinclude, prior to step 2, step 1 of coupling the compound (a4) with thecompound (b4) and obtaining the compound (c4).

The reaction of above step 1 can be performed under Wittig reactionconditions. This reaction can be performed under an inert gas atmospheresuch as argon or nitrogen. This reaction preferably uses a base such assodium hydride, sodium methoxide, or sodium ethoxide. This reaction ispreferably performed in an inert solvent such as tetrahydrofuran (THF)or N,N-dimethylformamide (DMF). The temperature of this reaction is notlimited, but can be in a range from 0° C. (in an ice bath) to roomtemperature.

The reaction of above step 2 is performed under Boc (tert-butoxycarbonylgroup) deprotection conditions. Lewis acid is preferably BB_(r3). Thisreaction can be performed under an inert gas atmosphere such as argon ornitrogen. This reaction can be performed in an inert solvent such asdichloromethane or chloroform. The temperature of this reaction can bemade room temperature.

If necessary, it is possible to protect each compound with an adequateprotecting group prior to the reactions of above step 1 and/or step 2,and then perform the reactions. When one or more of R₁, R₃ to R₅ have ahydroxy or an amino group, it is preferable to protect that hydroxy oramino group with an adequate protecting group. Examples of protectinggroups for hydroxy or amino groups include alkyl groups such as methylgroups and ethyl groups, benzyl groups, t-butyldimethylsilyl groups(—Si(CH₃)₂(t-C₄H₉)), tert-butoxycarbonyl groups (Boc: —COO-(t-C₄H₉)),methoxymethyl groups (—CH₂OCH₃), and ethoxymethyl groups (—CH₂OCH₂CH₃).Deprotection may be performed in adequate steps, in a method that isknown to one of skill in the art.

Synthesis Example 5

The compound of the present invention according to the formula (I), inwhich R₁ and R₂ are not hydrogen, can be prepared according to followingscheme 5.

In the above formulas, A, B, R₁ to R₅, and

[Formula 32]

have been defined above in the compound of the formula (I), in which,however, R₁ and R₂ are not hydrogen, X is an elimination group, whichis, for example, halogen such as Cl, Br or I, alkoxy such as methoxy orethoxy, triflate (—OSO₂—CF₃), carboxylate (—OCO—R), or an azide group(—N₃).

The compound of the formula (I-ii), which is the starting substance, canbe synthesized in accordance with above scheme 3 or 4. With the methodof preparing the compounds of the present invention, it is possible toinclude step 1 of obtaining the compound of the formula (I-iii) (thecompound of the formula (I), in which R₁ and R₂#H) by reacting thecompound of the formula (I-ii) with R₂X with reference to above scheme5. When R₁ and R₂ are the same group, it is possible to synthesize thecompound of the formula (I-iii) directly from the compound of theformula (I-i), in above scheme 3 or 4.

Similar to the reaction in step 1 of scheme 3 above, the reaction instep 1 of scheme 5 is alkylation, alkenylation, acylation orhydroxyalkylation of an amino group. Step 1 of scheme 5 can be performedunder the same conditions as for step 1 of above scheme 3.

If necessary, it is possible to protect each compound with an adequateprotecting group prior to the reaction of above step 1, and then performthe reaction. When one of more of R₁ to R₅ have a hydroxy or an aminogroup, it is preferable to protect that hydroxy or amino group with anadequate protecting group. Examples of protecting groups for hydroxy oramino group include alkyl groups such as methyl groups and ethyl groups,benzyl groups, t-butyldimethylsilyl groups (—Si(CH₃)₂(t-C₄H₉)),tert-butoxycarbonyl groups (Boc: —COO-(t-C₄H₉)), methoxymethyl groups(—CH₂OCH₃), and ethoxymethyl groups (—CH₂OCH₂CH₃). Deprotection may beperformed in a method that is known to one of skill in the art.

For example, when one or both of R4 and R5 are OH, the formula (I-ii)protector may be prepared as shown in step 2 of scheme 5. Step 2 shownin scheme 5 is a step for when R₄ alone is OH. This protector can beobtained by reacting the compound of the formula (I-ii) witht-butyldimethylchlorosilane. This reaction may use a base such asimidazole. This reaction is preferably performed under an inert gasatmosphere such as nitrogen or argon. Also, this reaction is usuallyperformed under an inert solvent such as dimethylsulfoxide. Thetemperature of this reaction is preferably room temperature. One ofskill in the art should readily understand that protector can besynthesized when R₄ is not OH and R₅ is OH, as well as when R₄ and R₅are OH.

After the formula (I-ii) protector is prepared, it is possible toprepare the formula (I-iii) protector by a reaction with R₂X, (Step 3).This reaction may adopt the same reaction conditions as in step 1 above.After that, by deprotecting the formula (I-ii) protector, the compoundof the formula (I-iii), in which one or both of R4 and R5 are OH, can beobtained. This deprotection can be performed using acid such ashydrochloric acid, or fluoride ion.

In above step 1, when R₂X is [¹¹C]alkyl-X such as ¹¹CH₃—X, aradioisotope for [¹¹C] alkyl, such as —¹¹CH₃, can be introduced.

Synthesis Example 6

The compound of the present invention according to the formula (I), inwhich R₃ is halogen, can be prepared according to following scheme 6:

In the above formulas, A, B, R₁ to H₅, and

[Formula 34]

have been defined above in the compound of the formula (I), in which,however, R₃ is halogen, especially F. With the method of scheme 6, aradioisotope for ¹⁸F can be introduced.

If necessary, it is possible to protect each compound with an adequateprotecting group prior to the reaction of above step 1, and then performthe reaction. When one or more of R₁ to R₅ have a hydroxy or an aminogroup, it is preferable to protect that hydroxy or amino group with anadequate protecting group. Examples of protecting groups for hydroxy oramino groups include alkyl groups such as methyl groups and ethylgroups, benzyl groups, t-butyldimethylsilyl groups (—Si(CH₃)₂(t-C₄H₉)),tert-butoxycarbonyl groups (Boc: —OOO-(t-C₄H₉)), methoxymethyl groups(—CH₂OCH₃), and ethoxymethyl groups (—CH₂OCH₂CH₃). Deprotection may beperformed in a method that is known to one of skill in the art.

For example, when, as shown in step 2, one or both of R₁ and R₂ arehydrogen, it is preferable to protect with a protecting group such as atert-butoxycarbonyl group (Boc: —COO-(t-C₄H₉)), prior to the reaction ofstep 1. Also, when one or both of R₄ and R₅ are OH, it is preferable toprotect with an ethoxymethyl group (—CH₂OCH₂CH₃), prior to the reactionof step 1.

Synthesis Example 7

The compound of the present invention according to the formula (I), inwhich R₄ is alkoxy, may be prepared according to following scheme 7.

In the above formulas, A, B, R₁ to R₃, R₅, and

[Formula 36]

have been defined above in the compound of the formula (I), in which Alkmeans alkyl and X means an elimination group.

With reference to above scheme 7, the method of preparing the compoundsof the present invention can include step 1 of obtaining the compound ofthe formula (I-v) (the compound of the formula (I), in which R₄ ismethoxy), by reacting the compound (a7) with Alk-X.

If necessary, it is possible to protect each compound with an adequateprotecting group prior to the reaction of above step 1, and then performthe reaction. When one or more of R₁ to R₃, and R₅ have a hydroxy or anamino group, it is preferable to protect this hydroxy or amino groupwith an adequate protecting group. Examples of protecting groups forhydroxy or amino groups include alkyl groups such as methyl groups andethyl groups, benzyl groups, t-butyldimethylsilyl groups(—Si(CH₃)₂(t-C₄H₉)), tert-butoxycarbonyl groups (Boc: —COO-(t-C₄H₉)),methoxymethyl groups (—CH₂OCH₃), and ethoxymethyl groups (—CH₂OCH₂CH₃).Deprotection may be performed in a method that is known to one of skillin the art.

When Alk-X is [¹¹C]alkyl-X such as ¹¹CH₃—X, a radioisotope for[¹¹C]alkyl, such as —¹¹CH₃, can be introduced.

Synthesis Example 8

The compound of the present invention according to the formula (I), inwhich R₄ is halohydroxyalkoxy, may be prepared according to followingscheme 8.

In the above formulas, A, B, R₁ to R₃, R₅, and

[Formula 38]

have been defined above in the compound of the formula (I), in which Alkmeans an alkyl group, TsO means tosylate (p-H₃C—C₆H₄—SO₂—O—), and Halmeans halogen, especially F.

With reference to above scheme 8, the method of preparing the compoundsof the present invention can include step 1 of obtaining the compound ofthe formula (I-v) (the compound of the formula (I), in which R₄ ismethoxy) by reacting the compound (a8-1) or (a8-2).

In the above compounds (a8-1) and (a8-2),

mean, respectively, a group in which TsO- and -0-2-tetrahydropyranyl arebound to given positions of the carbon atoms of —O-alkyl (alkoxy), and agroup in which TsO- and OH are bound to given positions of the carbonatoms of —O-alkyl (alkoxy). For example, the above formulas mean—O—CH₂CH (—O-2-tetrahydropyranyl) (—CH₂—OTs) or —O—CH₂CH (—OH)(—CH₂—OTs), and —O—CH (—O-2-tetrahydropyranyl) (—CH₂—OTs) or —O—CH(—CH₂—OH) (—CH₂-OTs), and so on.

Similarly, in the above formulas,

means a group in which Hal and OH are bound to given positions of thecarbon atoms of —O-alkyl(alkoxy), that is, halohydroxyalkoxy.

Synthesis Example 9

The compound of the present invention according to the formula (I), inwhich

is a triple bond, may be prepared according to following scheme 9.

In the above formulas, A, B, and R₁ to R₅ have been defined above withthe compound of the formula (I).

With reference to above scheme 9, the method of preparing the compoundsof the present invention can include step 1 of obtaining the compound ofthe formula (I-vi) (the compound of the formula (I), in which

is a triple bond) by coupling the compound (a9) with the compound (b9).

The reaction of above step 1 is performed under Sonogashira reactionconditions. This reaction can be performed using a copper catalyst suchas cuprous iodide, a palladium catalyst such as dichlorobis(triphenylphosphine) palladium, and a base such as triethylamine. Thetemperature of this reaction may be 25° C. to 120° C., preferably 50° to1000, and most preferably 70°.

If necessary, it is possible to protect each compound with an adequateprotecting group prior to the reaction of above step 1, and then performthe reaction. When one or more of R₁ to R₅ have a hydroxy or an aminogroup, it is preferable to protect that hydroxy or amino group with anadequate protecting group. Examples of protecting groups for hydroxy oramino groups include alkyl groups such as methyl groups and ethylgroups, benzyl groups, t-butyldimethylsilyl groups (—Si(CH₃)₂(t-C₄H₉)),tert-butoxycarbonyl groups (Boc: —COO-(t-C₄H₉)), methoxymethyl groups(—CH₂OCH₃), and ethoxymethyl groups (—CH₂OCH₂CH₃). Deprotection may beperformed in a method that is known to one of skill in the art.

4. Intermediates

The present invention provides an intermediate for synthesizing thecompound of the present invention according to the formula (I).Preferably, the intermediate is selected from the group consisting ofthe following:

the formula (I-i) in above scheme 2 (in the formula, R₄ is hydroxy);

the formula (I-ii), the formula (I-i) protector 1, and the formula (I-i)protector 2 in above scheme 3;

the formula (I-ii) protector in above scheme 5;

(a6) and the (a6) protector in above scheme 6;

the (a7) protector in above scheme 7; and

(a8-1) and (a8-2) in above scheme 8.

In one embodiment, the present invention provides an intermediate forsynthesizing the compound of the present invention according to theformula (I), selected from the group consisting of the following:

TABLE 2 Syn- thesis Embod- Name Name of Compound Structural Formulaiment (pro2) Synthetic intermediate of PBB2 2-((1E,3E)-4-(4-aminophenyl) buta-1,3-dienyl)benz[d] thiazole-6-ol

30 (pro3) Synthetic intermediate of PBB3 5-((1E,3E)-4-(6-(tert-butyldimethylsilyloxy) benz[d]thiazole-2- yl)buta-1,3-dienyl)pyridine-2-amine

31 (pro6) Synthetic intermediate of mPBB5 2-((1E,3E)-4-(4-(dimethylamino) phenyl)buta- 1,3-dienyl)-3-ethyl-6- hydroxy-benz[d]thiazole-3-ium

32 (pro7) Synthetic intermediate of PBB2.1 (E)-2-(4-(4- methylamino)phenyl)buta- 1-en-3-ynyl) benz[d]thiazole-6-ol

 7 (pro8) Synthetic intermediate of PBB2.2 (E)-2-(4-(4- aminophenyl)buta-1-en-3- ynyl)benz[d] thiazole-6-ol

 8 (pro11) Synthetic intermediate of PBB3.2 (E)-5-(4-(6-(tert-butyldimethylsilyloxy) benz[d]thiazole-2-yl) buta-3-en-1-ynyl)pyridine-2- amine

34 (pro1

) Synthetic intermediate of PBB3.2N (E)-tert-butyl(2-(4-(6-aminopyridine-3-yl) buta-1-en-3-ynyl) benz[d]thiazole-6-yl)methylcarbamate

34 Synthetic intermediate of F0-FBB3 analog 2-(2-((1E,3E)-4-(6-(dimethylamino) pyridine-3-yl)buta- 1,3-dienyl)benzo[d]thiazole-6-yloxy)-2- hydroxymethyl-ethyl 4- methylbenzenesulfonate

35-1 (pro21) Synthetic intermediate of F0-FBB3 3-(2-((1E,3E)-4-(6-(methylamino) pyridine-3-yl) buta-1,3-dienyl) benzo[d]thiazole-6-yloxy)- 2-(tetrahydro- 2H-pyran-2-yloxy)propyl 4-methylbenzenesulfonate

35-2 (pro22) Synthetic intermediate of F0-FBB3.2 (E)-3-(2-(4-(6-(methylamino) pyridine-3-yl)buta-1- en-3-ynyl)benz[d] thiazole-6-yloxy)-2-(tetrahydro-2H- pyran-2-yloxy)propyl 4- methylbenzenesulfonate

36 (pro23) Synthetic intermediate of F1-FBB3 Tert-butyl 5-((1E,3E)-4-(6-(ethoxymethoxy)benz[d] thiazole-2-yl)buta- 1,3-dienyl)-6-nitropyridine-2-yl (methyl)carbamate

37 (pro24) Synthetic intermediate of F1-FBB3.2 (E)-tert-butyl 5-(4-(6-(ethoxymethoxy)benz[d] thiazole-2-yl)buta-3-en-1-ynyl)-6-nitropyridine-2-yl (methyl)carbamate

38 (pro25) Synthetic intermediate of F1-FBB

3 Tert-butyl 5-((1E,3E)- 4-(5-(ethoxymethoxy) benzofuran-2-yl)buta-1,3-dienyl)-6- nitropyridine-2-yl (methyl)carbamate

39 (pro26) Synthetic intermediate of F1-FBB

3.2 (E)-tert-butyl 5-(4-(6- (ethoxymethoxy) benzofuran-2-yl)buta-3-en-1-ynyl)-6- nitropyridine-2-yl (methyl)carbamate

40

indicates data missing or illegible when filed

Preferably, the intermediate of the present invention is used tosynthesize the compound of the present invention according to theformula (I) labeled with a radioisotope.

5. Compositions

The present invention provides a composition for tau imaging, whichcontains the compound of the formula (I), or a pharmaceuticallyacceptable salt thereof or a solvate thereof. Also, this imagingincludes in vitro, ex vivo and in vivo imaging. The composition mayinclude a pharmaceutically acceptable carrier. Examples of thispharmaceutically acceptable carrier include water, saline water,physiological saline water or phosphate buffered saline water (PBS),sodium chloride injection solution, Ringer's injection solution,isotonic dextrose injection solution, sterile water injection solution,dextrose, and lactated Ringer's injection solution.

The contents of the compound of the formula (I) and the pharmaceuticallyacceptable carrier are not particularly limited, and these aredetermined based on various factors such as: the type of the compoundthat is used: the age, weight, health conditions, sex, and content ofdiet of the mammals that receive an administration; the number ofadministration and the route of administration; the period of treatment;other medicines that are at the same time, and so on. The content of thepharmaceutically acceptable carrier may be made an amount of 1 to 99weight % of the composition of the present invention. The composition ofthe present invention may preferably be adjusted such that the compoundof the formula (I) can be administered in an amount of 0.01 mg/kg to 5mg/kg, or 0.05 mg/kg to 3 mg/kg, and preferably 0.1 mg/kg to 1 mg/kg.

6. Methods of Use of the Compounds of the Present Invention

The compounds of the present invention can be used as a molecular probefor tau imaging, that is, as a molecular probe for imaging tau proteinsthat accumulate in the brains of mammals. Consequently, the presentinvention provides a tau imaging method that includes administering thecompound of the formula (I), or a pharmaceutically acceptable salt or asolvate thereof, to mammals. In another embodiment, the presentinvention provides a tau imaging method that includes (a) a step ofadministering an effective dose of the compound of the formula (I), or apharmaceutically acceptable salt or a solvate thereof, to a mammal, and(b) a step of imaging the brain of the mammal.

The mammal may be, for example, a human, rat, mouse, rabbit, guinea pig,hamster, monkey, dog, ferret or miniature swine. Preferably, the mammalis a human. The method of administration is not particularly limited,and, for example, parenteral administration, intravenous administration,or intraperitoneal administration may be used. Preferably, intravenousadministration or intraperitoneal administration may be used. Mostpreferably, intravenous administration may be used. The amount ofadministration is preferably 0.01 mg/kg to 5 mg/kg, 0.05 mg/kg to 3mg/kg, or 0.1 mg/kg to 1 mg/kg, and most preferably 0.1 mg/kg to 1mg/kg.

This imaging can be performed by molecular imaging methods such aspositron emission tomography (PET), fluorescence microscopy measurement,multi-photon imaging, two-photon imaging, near-infrared fluorescenceimaging, autoradiography, and single-photon emission computed tomography(SPECT). Also, this imaging includes in vitro, ex vivo, and in vivoimaging.

In one embodiment, the present invention provides a composition, whichis for diagnosing diseases that are caused by accumulation of tauproteins in the brain, and which contains the compound of the formula(I), or a pharmaceutically acceptable salt or a solvate thereof. Inanother embodiment, the present invention provides a method ofdiagnosing diseases that are caused by accumulation of tau proteins,including administering the compound of the formula (I), or apharmaceutically acceptable salt or a solvate thereof, to a mammal.

Diseases that are caused by accumulation of tau proteins include, forexample, Alzheimer's disease, non-Alzheimer-type tauopathies (includingfrontotemporal lobar degeneration), or other tau-positiveneurodegenerative diseases. To be more specific, diseases that arecaused by accumulation of tau proteins include, besides Alzheimer'sdisease, progressive supranuclear palsy (PSP), Pick's disease,corticobasal degeneration (CBD), frontotemporal lobar degeneration(FTLD), frontotemporal dementia with Parkinsonism linked to chromosome17 (FTDP-17), argyrophilic grain disease (AGD), dementiapugilistica-boxer's encephalopathy, Parkinson-dementia complex of Guam,or neurofibrillary tangle-predominant dementia.

In another embodiment, the present invention provides a method ofdiagnosing a disease that is caused by accumulation of tau proteins, andthis diagnosis method includes (a) a step of administering an effectivedose of the compound of the formula (I), or a pharmaceuticallyacceptable salt or a solvate thereof, to a mammal, and (b) a step ofimaging the brain of the mammal. In another embodiment, the above methodfurther includes (c) a step of comparing the image of the brain of themammal with that of a normal mammal. If the fluorescence intensityand/or the radiation intensity of the compound of the present inventionshow an increase compared to the normal mammal, this indicates that adisease to be caused by accumulation of tau proteins might havedeveloped or that there is a danger of developing it.

In yet another embodiment, the present invention provides use of thecompound of the formula (I), or a pharmaceutically acceptable salt or asolvate thereof, for preparing a composition for tau imaging. In yetanother embodiment, the present invention provides use of the compoundof the formula (I), or a pharmaceutically acceptable salt or a solvatethereof, for preparing a composition for diagnosing diseases such asAlzheimer's disease, frontotemporal lobar degeneration, dementia, orother neurodegenerative tauopathies.

In one embodiment, the present invention provides a method of screeninga compound for treating or preventing a disease or a symptom that iscaused by accumulation of tau proteins in the brain, and this screeningmethod includes (a) a step of administering, to a mammal having adisease or a symptom that is caused by accumulation of tau proteins, acandidate compound for treating or preventing the disease or symptom,(b) a step of administering an effective dose of the compound of theformula (I) or a pharmaceutically acceptable salt thereof, to themammal, and (c) a step of imaging the brain of the mammal.

The above screening method can further include (d-1) a step of comparingthe image of the brain of the mammal with that from before theadministration of the candidate compound. If, after the candidatecompound is administered, the fluorescence intensity and/or theradiation intensity of the compound of the present invention show adecrease compared to those from before the administration of thecandidate compound, this indicates that the candidate compound iseffective as a compound for treating the disease or the symptom.Alternatively, the above method can include (d-2) a step of comparingthe image of the brain of the mammal with an image of another normalmammal. If, after the candidate compound is administered, thefluorescence intensity and/or the radiation intensity of the compound ofthe present invention are equal to those of the normal mammal, thisindicates that the candidate compound is effective as a compound fortreating the disease or the symptom.

EMBODIMENTS 7. Embodiments

Embodiments of the present invention will be described below. Theseembodiments will be described only to deepen the understanding of theclaims of the present invention, and are by no means intended to limitthe claims of the present invention.

Synthesis of the Compounds of the Present Invention Synthesis Embodiment1 Synthesis of4-((1E,3E)-4(benz[d]thiazole-2-yl)buta-1,3-dienyl)-N,N-dimethylaniline(PBB1)

PBB1 was synthesized according to the following synthesis scheme.

2-(bromomethyl)benzothiazole (Wako Code: Alfa Aesar, H26120) was reactedwith trimethyl phosphite (Wako Code: 200-09082, 204-09085), and theresulting product was reacted with p-(dimethylamino)cinnamaldehyde (WakoCode: 045-16441, 041-16443, 043-16442), and the target compound wasobtained.

PBB1: ¹H NMR (300 MHz, DMSO-d₆) δ ppm: 8.04 (d, J=7.80 Hz, 1H), 7.90 (d,J=7.80 Hz, 1H), 7.48 (dd, J=7.80 Hz, 7.80 Hz, 1H), 7.36-7.43 (m, 4H),6.89-6.98 (m, 3H), 6.72 (d, J=8.7 Hz, 2H), 2.96 (s, 6H)

Synthesis Embodiment 2 Synthesis of2-((1E,3E)-(4-(methylamino)phenyl)buta-1,3-dienyl)benz[d]thiazole-6-ol(PBB2)

PBB2 was synthesized according to the following synthesis scheme.

Step 1: Synthesis of Carboxylic Acidtert-butylester2-methyl-benzothiazole-6-ylester (1)

Triethylamine (6.58 ml, 47.5 mmol) and an anhydrous dimethylformamidesolution (48 ml) of di-tert-butyl dicarbonate (10.8 g, 49.5 mmol) wereadded in an anhydrous dimethylformamide solution (150 ml) of2-methyl-benzothiazole-6-ol (3.27 g, 19.8 mmol). The reaction mixturewas stirred for 16 hours. The reaction mixture was condensed, and theresidue was refined by column chromatography (ethyl acetate/hexane=1:3).The desired product was obtained as a pale brown solid, at a yield of99% (5.23 g).

¹H NMR (400 MHz, CDCl₃) δ ppm 7.91 (d, J=8.8 Hz, 1H), 7.66 (d, J=2.3 Hz,1H), 7.25 (dd, J=8.8, 2.4 Hz, 1H), 2.82 (s, 3H), 1.57 (s, 9H).

Step 2: Synthesis of Carboxylic Acid2-{4-[4-(tert-butoxycarbonyl-methyl-amino)-phenyl]-buta-1,3-dienyl}-benzothiazole-6-ylestertert-butylester(2)

Finely powdered sodium hydroxide (892 mg, 22.3 mmol) was added in adimethylformamide solution (15 ml) of carboxylic acidtert-butylester2-methyl-benzothiazole-6-ylester (1) (947 mg, 3.57 mmol).The solution was stirred for 10 minutes, and, after that, adimethylformamide solution (6.2 ml) of 4-N-Boc-4-N-methyl-cinnamaldehyde(933 mg, 3.57 mmol) was added dropwise. The reaction mixture was stirredfor 3.5 hours. The reaction mixture was diluted with ethyl acetate, andwas washed with water. The aqueous phase was extracted 5 times usingethyl acetate. The combined ethyl acetate phase was dried with sodiumsulfate and condensed. The residue was refined by column chromatography(ethyl acetate/hexane=1:3→1:2). The desired product was obtained as abright yellow solid, at a yield of 76% (1.12 g).

¹H NMR (400 MHz, CDCl₃) δ ppm 8.33 (bs, 1H), 7.67 (d, J=8.8 Hz, 1H),7.27 (d, J=8.5, Hz, 2H), 7.18 (d, J=8.5 Hz, 2H), 7.08 (dd, J=15.4, 10.5Hz, 1H), 7.04 (bs, 1H), 6.84 (d, J=15.4 Hz, 1H), 6.90-6.78 (m, 1H), 6.71(dd, J=15.2, 10.5 Hz, 1H), 6.61 (d, J=15.5 Hz, 1H), 3.26 (s, 3H), 1.51(s, 9H).

(Note that 4-N-Boc-4-N-methyl-cinnamaldehyde was synthesized accordingto the method disclosed in Japanese Unexamined Patent ApplicationPublication No. 2007-106755).

Step 3: Synthesis of2-[4-(4-methylamino-phenyl)-buta-1,3-dienyl]-benzothiazole-6-ol (3)

Carboxylic acid2-{4-[4-(tert-butoxycarbonyl-methyl-amino)-phenyl]-buta-1,3-dienyl}-benzothiazole-6-ylestertert-butylester(2) (1.07 g, 26.3 mmol) was suspended in dichloromethane (15.8 ml).Trifluoroacetic acid (15.8 ml) was added and the red solution wasstirred for 2 hours. The reaction mixture was condensed, and the residuewas dissolved in water. The solution was neutralized by addition of asaturated sodium hydrogen carbonate solution. The product wasprecipitated, and this was washed 3 times with water, and 3 times withdiethyl ether. The desired product was obtained as a red brown solid, ata yield of 72% (587 mg).

PBB2: ¹H NMR (400 MHz, DMF-d₇) δ ppm 9.56 (bs, 1H), 7.72 (d, J=8.7 Hz,1H), 7.39 (d, J=2.2 Hz, 1H), 7.37 (d, J=8.6, Hz, 2H), 7.28 (dd, J=15.5,8.9 Hz, 1H), 7.03 (dd, J=8.7, 2.0 Hz, 1H), 6.95-6.81 (m, 2H), 6.85 (d,J=15.4 Hz, 1H), 6.64 (d, J=8.4 Hz, 2H), 5.65 (bs, 1H), 2.83 (s, 3H)ESI-MS: m/z 309 [M−41]+

Synthesis Embodiment 3 Synthesis of2-((1E,3E)-4-(6-(methylamino)pyridine-3-yl)buta-1,3-dienyl)benz[d]thiazole-6-ol(PBB3)

PBB3 was synthesized according to the following synthesis scheme.

Step 1: Synthesis of Compound (10)

Under an argon atmosphere, 3,3-diethoxy-1-propene (58.58 g, 450.0 mmol),potassium chloride (11.18 g, 150.0 mmol), tetrabutylammonium and acetate(13.57 g, 45.0 mmol), potassium carbonate (31.10 g, 225.0 mmol) andpalladium acetate (1.68 g, 7.5 mmol) were added in aN,N-dimethylformamide solution (450 mL) of the compound (9) (30.45 g,150.0 mmol), heated to 100° C., and stirred all night. The reactionliquid was filtered and ethyl acetate and 1N hydrochloric acid wereadded thereto, and the reaction liquid was stirred. The reaction liquidwas neutralized by adding a sodium hydrogen carbonate aqueous solution,and the organic layer was extracted with ethyl acetate. After dryingwith anhydrous sodium sulphate, the solvent was distillated underreduced pressure. By refining the residue by column chromatography(developing solvent: chloroform), 3.31 g of the title compound (10) wasobtained.

Step 2: Synthesis of Compound (11)

Under an argon atmosphere, after a tetrahydrofuran solution (166 mL) ofthe compound (6) (5.98 g, 18.96 mmol) was cooled with ice, sodiumhydride (60% oil, 758 mg, 18.96 mmol) was added. The reaction liquid washeated to room temperature, and, after stirring for 30 minutes, thecompound (10) (2.94 g, 16.50 mmol) was added. After the disappearance ofthe raw material, the reaction liquid was added in water and stirred,and the precipitate was filtered. Toluene was added to the cake, and thesolvent was distillated under reduced pressure, and suspended and washedwith toluene. The precipitate was filtered and dried under reducedpressure, thereby giving 4.06 g of the title compound (11).

Step 3: Synthesis of Compound (12)

Acetic acid (76 mL), iron (3.06 g, 54.79 mmol) and 12N hydrochloric acid(16 mL) were added in an ethanol solution (76 mL) of the compound (11)(3.96 g, 11.67 mmol), and the resultant solution was stirred all night.The reaction liquid was added dropwise in a sodium hydroxide aqueoussolution under ice cold conditions, and the precipitate was filtered.Methanol was added to the cake, and the resultant mixture was stirredand filtered. By distillating the filtrate under reduced pressure andrefining the residue by column chromatography (developing solvent:chloroform→chloroform/methanol=20/1), 1.29 g of the title compound (12)was obtained.

Step 4: Synthesis of Compound (13)

Under an argon atmosphere, after a N,N-dimethylformamide solution (21mL) of the compound (12) (1284 mg, 4.15 mmol) was cooled with ice,sodium hydride (60% oil, 183 mg, 4.57 mmol) was added. The reactionliquid was heated to room temperature, and, after stirring for 30minutes, methyl iodide (556 mg, 3.92 mmol) was added. The reactionliquid was added in water and stirred, and extracted with chloroform.The organic layer was washed with saturated saline water, and, afterdrying with anhydrous sodium sulphate, the solvent was distillated underreduced pressure. By refining the residue by column chromatography(developing solvent: chloroform→chloroform/methanol=97/3), 188 mg of thetitle compound (13) was obtained.

Step 5: Synthesis of2-((1E,3E-4-(6-(methylamino)pyridine-3-yl)buta-1,3-dienyl)benz[d]thiazole-6-ol(PBB3)

Under an argon atmosphere, after a dichloromethane solution (2.9 mL) ofthe compound (13) (184 mg, 0.57 mmol) was cooled down to −78° C., borontribromide (1.0 M dichloromethane solution, 2.85 mL, 2.85 mmol) wasadded dropwise. The reaction liquid was heated to room temperature, andstirred all night. After the reaction liquid was neutralized by adding a1N sodium hydroxide aqueous solution and sodium hydrogen carbonate underice cold conditions, the precipitate was filtered. The cake was washedwith water and diethyl ether, and after methanol was added thereto andthe resultant mixture was stirred, the resultant mixture was filtered.After the filtrate was distillated under reduced pressure, 120 mg of thetitle compound was obtained by refining the residue by columnchromatography (developing solvent: chloroform/methanol=97/3→19/1).

PBB3: ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 9.83 (s, 1H), 8.09 (d, J=2.29 Hz,1H), 7.71 (d, J=8.70 Hz, 1H), 7.69 (dd, J=9.16 Hz, 2.29 Hz, 1H), 7.32(d, J=2.75 Hz, 1H), 7.22 (dd, J=15.57 Hz, 10.53 Hz, 1H), 6.87-7.00 (m,3H), 6.84 (d, J=15.57 Hz, 1H), 6.83 (d, J=15.11 Hz, 1H), 6.48 (d, J=8.70Hz, 1H), 2.80 (d, J=5.04 Hz, 3H)

Synthesis Embodiment 4 Synthesis of 2-((1E,3E)-4-(6-(methylamino)pyridine-3-yl)buta-1,3-dienyl)benz[d]thiazole-5,6-diol(PBB4)

PBB4 was synthesized according to the following synthesis scheme.

Step 1: Synthesis of6-tert-butoxycarbonyloxy-2-methyl-benzothiazole-5-ylestertert-butylester(4)

Triethylamine (23.2 ml, 172 mmol), an anhydrous dimethylformamidesolution (48 ml) of di-tert-butyl dicarbonate (37.4 g, 172 mmol), and4-dimethylaminopyridine (838 mg, 6.86 mmol) were added in an anhydrousdimethylformamide solution (260 ml) of 2-methyl-benzothiazole-5,6-diol(6.22 g, 34.3 mmol). The reaction mixture was stirred for 4 hours. Thereaction mixture was condensed, and the residue was refined by columnchromatography (ethyl acetate/hexane=1:4). The desired product wasobtained as a pale brown solid, at a yield of 93% (12.26 g).

¹H NMR (400 MHz, CDCl₃) δ ppm 7.81 (s, 1H), 7.72 (s, 1H), 2.82 (s, 3H),1.564 (s, 9H), 1.558 (s, 9H).

Step 2: Synthesis of{4-[4-(5,6-dihydroxy-benzothiazole-2-yl)-buta-1,3-dienyl]-phenyl}-methyl-carbamicAcid tert-butylester (5)

Finely powdered sodium hydroxide (1.42 g, 35.6 mmol) was added in adimethylformamide solution (30 ml) of6-tert-butoxycarbonyloxy-2-methyl-benzothiazole-5-ylestertert-butylester(4) (2.17 g, 5.7 mmol). The solution was stirred for 10 minutes, and,after that, a dimethylformamide solution (4.2 ml) of4-N-Boc-4-N-methyl-cinnamaldehyde/cinnamaldehyde (1.5 g, 5.74 mmol) wasadded dropwise. The reaction mixture was stirred for 4.5 hours. Thereaction mixture was diluted with ethyl acetate, and was washed withwater. The aqueous phase was extracted 5 times using ethyl acetate. Thecombined ethyl acetate phase was dried with sodium sulfate, andcondensed. The residue was refined by column chromatography (ethylacetate/hexane=1:1). The desired product was obtained as anorange-yellow solid at a yield of 27% (667 mg).

¹H NMR (400 MHz, DMSO-D6) δ ppm 9.51 (bs, 1H), 9.42 (bs, 1H), 7.51 (d,J=8.5, Hz, 2H), 7.29 (d, J=8.3, Hz, 2H), 7.285 (s, 1H), 7.26 (s, 1H),7.23-7.10 (m, 2H), 6.95 (d, J=15.1 Hz, 1H), 6.94 (d, J=15.1 Hz, 1H),3.19 (s, 3H), 1.40 (s, 9H).

Step 3: Synthesis of2-[4-(4-methylamino-phenyl)-buta-1,3-dienyl]-benzothiazole-5,6-diol (6)

{4-[4-(5,6-dihydroxybenzothiazole-2-yl)-buta-1,3-dienyl]-phenyl}-methyl-carbamicacid tert-butylester (5) (614 mg, 1.45 mmol) was suspended indichloromethane (8 ml). Trifluoroacetic acid (8 ml) was added, and thered solution was stirred for 2 hours. The reaction mixture wascondensed, and the residue was dissolved in water. The solution wasneutralized by addition of a saturated sodium hydrogen carbonatesolution. The product was precipitated, and this was washed 3 times withwater, and 3 times with diethyl ether. The desired product was obtainedas a brown solid, at a yield of 58% (276 mg).

PBB4: ¹H NMR (400 MHz, DMF-d₇) δ ppm 9.60 (bs, 2H), 7.52-7.29 (m, 4H),7.27 (dd, J=15.2, 10.6 Hz, 1H), 6.96 (dd, J=15.2, 10.3 Hz, 1H),6.91-6.81 (m, 2H), 6.63 (d, J=8.1 Hz, 2H), 6.06 (d, J=4.1 Hz, 1H), 2.81(d, J=4.3 Hz, 3H). ESI-MS: m/z 325 [M+H]+

Synthesis Embodiment 5 Synthesis of2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dienyl)-3-ethyl-6-methoxybenzo[d]thiazole-3-ium(mPBB5)

The synthesis was performed by a method that was similar to thesynthesis method of PBB5.

Synthesis Embodiment 6 Synthesis of(E)-2-(4-(4-(dimethylamino)phenyl)buta-1-en-3-ynyl)benz[d]thiazole-6-ol(PBB2.1)

The synthesis was performed by a method similar to that of followingsynthesis example 10.

Synthesis Embodiment 7 Synthesis of(E)-2-(4-(4-(methylamino)phenyl)buta-1-en-3-ynyl)benz[d]thiazole-6-ol(PBB2.2)

The synthesis was performed by a method similar to that of followingsynthesis example 10.

Synthesis Embodiment 8 Synthesis of(E)-2-(4-(4-aminophenyl)buta-1-en-3-ynyl)benz[d]thiazole-6-ol (PBB2.3)

The synthesis was performed by a method similar to that of followingsynthesis example 10.

Synthesis Embodiment 9 Synthesis ofE)-2-(4-(6-(dimethylamino)pyridine-3-yl)buta-1-en-3-ynyl)benz[d]thiazole-6-ol(PBB3.1)

The synthesis was performed by a method similar to that of followingsynthesis example 10.

Synthesis Embodiment 10 Synthesis of(E)-2-(4-(6-(methylamino)pyridine-3-yl)buta-1-en-3-ynyl)benz[d]thiazole-6-ol(PBB3.2)

PBB3.2 was synthesized according to the following synthesis scheme.

Step 1: Synthesis of Compound (16)

Under an argon atmosphere, after a N,N-dimethylformamide solution (2.9mL) of 2-(t-butoxycarbonylamino)-5-iodopyridine (15) (640 mg, 2.00 mmol)was cooled with ice, cesium carbonate (1088 mg, 3.34 mmol) and methyliodide (497 mg, 3.50 mmol) were added, and the resultant solution wasstirred. After the disappearance of the raw material was confirmed,water was added in the reaction liquid, and the organic layer wasextracted using ethyl acetate. The organic layer was washed with waterand saturated saline water, and, after drying with anhydrous sodiumsulphate, the solvent was distillated under reduced pressure. Byrefining the residue by column chromatography (developing solvent:heptane/ethyl acetate=50/1→heptane/ethyl acetate=10/1), 575 mg of thetitle compound (16) was obtained.

Step 2: Synthesis of Compound (17)

Under an argon atmosphere, copper iodide (39 mg, 0.20 mmol),2-propyn-1-ol (191 mg, 3.41 mmol) and dichlorobis (triphenylphosphine)palladium (II) (24 mg, 0.03 mmol) were added in a triethylamine solution(1.66 mL, 11.90 mmol) of the compound (16) (568 mg, 1.70 mmol), and theresultant solution was stirred. After the disappearance of the rawmaterial was confirmed, water was added in the reaction liquid, and theorganic layer was extracted using ethyl acetate. The organic layer waswashed with water and saturated saline water, and, after drying withanhydrous sodium sulphate, the solvent was distillated under reducedpressure. By refining the residue by column chromatography (developingsolvent: heptane/ethyl acetate=4/1→heptane/ethyl acetate=3/2), 400 mg ofthe title compound (17) was obtained.

Step 3: Synthesis of Compound (18)

Under an argon atmosphere, triethylamine (501 mg, 4.95 mmol) and apyridine sulfur trioxide complex (716 mg, 4.50 mmol) were added in adimethylsulfoxide solution (7.50 mL) of the compound (17) (393 mg, 1.50mmol), and the resultant solution was stirred. After the disappearanceof the raw material was confirmed, water was added in the reactionliquid, and the organic layer was extracted using ethyl acetate. Theorganic layer was washed with water and saturated saline water, and,after drying with anhydrous sodium sulphate, the solvent was distillatedunder reduced pressure. By refining the residue by column chromatography(developing solvent: heptane/ethyl acetate=20/1→heptane/ethylacetate=10/1), 315 mg of the title compound (18) was obtained.

Step 4: Synthesis of Compound (19)

Under an argon atmosphere, after a tetrahydrofuran solution (10 mL) ofthe compound (6) (315 mg, 1.00 mmol) was cooled with ice, sodium hydride(60% oil, 48 mg, 1.20 mmol) was added. After the reaction liquid washeated to room temperature and stirred for 30 minutes, the compound (18)(312 mg, 1.20 mmol) was added. After the disappearance of the rawmaterial, water was added in the reaction liquid, and the organic layerwas extracted using ethyl acetate. The organic layer was washed withwater and saturated saline water, and, after drying with anhydroussodium sulphate, the solvent was distillated under reduced pressure. Byrefining the residue by column chromatography (developing solvent:heptane/ethyl acetate=10/1→heptane/ethyl acetate=5/1), 340 mg of thetitle compound (18) was obtained.

Step 5: Synthesis of(E)-2-(4-(6-(methylamino)pyridine-3-yl)buta-1-en-3-ynyl)benz[d]thiazole-6-ol(PBB3.2)

Under an argon atmosphere, after a dichloromethane solution (4.0 mL) ofthe compound (18) (336 mg, 0.80 mmol) was cooled down to −50° C., borontribromide (1.0M dichloromethane solution, 6.38 mL, 6.38 mmol) was addeddropwise. The reaction liquid was heated to room temperature and stirredall night. After a 1N sodium hydroxide aqueous solution and sodiumhydrogen carbonate were added in the reaction liquid under ice coldconditions and neutralized, the precipitate was filtered. The cake waswashed with water and diisopropyl ether. After methanol was added andthe cake was stirred, the precipitate was filtered and dried underreduced pressure, thereby giving 130 mg of the title compound (4).

PBB3.2: ¹H NMR (400 MHz, DMSO-d6) δ ppm: 9.95 (s, 1H), 8.19 (d, J=2.29Hz, 1H), 7.78 (d, J=8.07 Hz, 1H), 7.48 (dd, J=8.70 Hz, 2.29 Hz, 1H),7.36 (d, J=2.29 Hz, 1H), 7.18 (d, J=16.03 Hz, 1H), 7.13 (q, J=4.58 Hz,1H), 6.97 (dd, J=8.70 Hz, 2.29 Hz, 1H), 6.85 (d, J=15.57 Hz, 1H), 6.48(d, J=8.07 Hz, 1H), 2.81 (d, J=4.58 Hz, 3H)

Synthesis Embodiment 11 Synthesis of(E)-5-(4-(6-(aminomethyl)benz[d]thiazole-2-yl)buta-3-en-1-ynyl)-N-methylpyridine-2-amine(PBB3.2N)

PBB3.2N was synthesized according to the following synthesis scheme.

Step 1: Synthesis of2-(N-(tert-butoxycarbonyl)-N-methylamino)-5-ethynylpyridine (CompoundA′)

Using 5-iodopyridine-2-amine as the starting substance, the synthesiswas performed with reference to literature that described the synthesismethods of a similar substance (N-tert-butoxycarbonylation andmethylation: WO2010/024769, ethynylation: C. B. Aarkeroy et al., DaltonTrans., 2006, 1627).

¹H-NMR (400 MHz, CDCl₃) δ ppm: 8.47 (d, J=2.0 Hz, 1H), 7.75 (d, J=8.8Hz, 1H), 7.68 (dd, J=8.8 Hz, 2.0 Hz, 1H), 3.41 (s, 3H), 3.15 (s, 1H),1.53 (s, 9H)

Note that 2-amino-5-ethynylpyridine, which was the starting substance,was synthesized with reference to literature (C. B. Aarkeroy et al.,Dalton Trans., 2006, 1627).

Step 2: Synthesis of 6-(bromomethyl)benzothiazole-2-carbonitrile (13)

In carbon tetrachloride (34 mL), 6-methylbenzothiazole-2-carbonitrile(CAS No. 39785-48-3) (1.18 g, 6.77 mmol), N-bromosuccinimide (1.22 g,6.85 mmol) and azobisisobutyronitrile (0.14 g, 0.85 mmol) were reactedfor 1 hour under reflux and then condensed under reduced pressure, theresidue was refined by silica gel column chromatography, and the titlecompound was obtained as a yellowish white solid (1.17 g, 4.62 mmol).

¹H-NMR (400 MHz, CDCl₃) δ ppm: 8.20 (d, J=8.4 Hz, 1H), 8.01 (d, J=1.6Hz, 1H), 7.68 (dd, J=8.4 Hz, 1.6 Hz, 1H), 4.64 (s, 2H)

Step 3: Synthesis ofN-(2-cyanobenzothiazole-6-ylmethyl)iminodicarboxylic Acidtert-butylmethyl(14)

A DMF solution (6 mL) of iminodicarboxylic acid tert-butylmethyl (0.48g, 2.8 mmol) was cooled with ice, 60% sodium hydride (0.11 g, 2.8 mmol)was added thereto, and the resultant solution was stirred for 30minutes. Next, a DMF solution (6 mL) of6-(bromomethyl)benzothiazole-2-carbonitrile (0.58 g, 2.3 mmol) wasadded, and the resultant mixture was stirred for 30 minutes at roomtemperature. The reaction mixture was added water and extracted withethyl acetate, the crude product was refined by silica gel columnchromatography, and the title compound was obtained as a liquid that wasvirtually colorless (0.71 g, 2.0 mmol).

¹H-NMR (400 MHz, CDCl₃) δ ppm: 8.17 (d, J=8.4 Hz, 1H), 7.92 (d, J=1.6Hz, 1H), 7.60 (dd, J=8.4 Hz, 1.6 Hz, 1H), 5.01 (s, 2H), 3.85 (s, 3H),1.45 (s, 9H)

Step 4: Synthesis of6-((tert-butoxycarbonylamino)methyl)benzothiazole-2-carboxylic AcidMethyl (16)

A 5M sodium hydroxide aqueous solution (2.05 mL, 10.25 mmol) was addedin a methanol solution (19 mL) ofN-(2-cyanobenzothiazole-6-ylmethyl)iminodicarboxylic acidtert-butylmethyl (0.71 g, 2.0 mmol), and the resultant solution wasstirred for 4 days at room temperature. After the solution wasneutralized with dilute hydrochloric acid, water was added thereto, theorganic layer was extracted with ethyl acetate, and the solvent waswashed with saturated saline water and dried with anhydrous sodiumsulphate. The solvent was distillated at reduced pressure, the residuewas dissolved in methanol (25 mL) and 1M hydrochloric acid (1.04 mL,1.04 mmol) was added thereto, and the resultant mixture was stirred for30 minutes at room temperature. Furthermore, after the mixture was added1M hydrochloric acid (1.04 mL, 1.04 mmol), stirred for 30 minutes atroom temperature and diluted with ethyl acetate, the resultant mixturewas washed with water, dried, and condensed at reduced pressure, and thetitle compound was obtained as a solid that was virtually white (0.62 g,2.0 mmol).

¹H-NMR (400 MHz, CDCl₃) δ ppm: 8.19 (d, J=8.4 Hz, 1H), 7.90 (s, 1H),7.50 (d, J=8.4 Hz, 1H), 5.0 (br, 1H), 4.49 (br d, J=5.2 Hz, 2H), 4.09(s, 3H), 1.48 (s, 9H)

Step 5: Synthesis of(6-((tert-butoxycarbonylamino)methyl)benzothiazole-2-yl)methanol (17)

Sodium borohydride (359 mg, 9.49 mmol) was added in a methanol solution(52 mL) of 6-((tertbutoxycarbonylamino)methyl)benzothiazole-2-carboxylic acid methyl (1.02g, 3.16 mmol), and the resultant solution was stirred at roomtemperature for 1 hour. Water was added to the reaction mixture, and theorganic layer was extracted with ethyl acetate and dried with anhydroussodium sulphate. The solvent was distillated at reduced pressure, andthe title compound was obtained as a pale yellowish white solid (0.93 g,3.16 mmol).

¹H-NMR (400 MHz, CDCl₃) δ ppm: 7.92 (d, J=8.4 Hz, 1H), 7.80 (s, 1H),7.38 (d, J=8.4 Hz, 1H), 5.07 (s, 2H), 5.0 (br, 1H), 4.44 (br d, J=6.0Hz, 2H), 2.97 (br, 1H), 1.47 (s, 9H)

Step 6: Synthesis of6-((tert-butoxycarbonylamino)methyl)benzothiazole-2-carboxaldehyde (18)

A Dess-Martin reagent (2.52 g, 5.94 mmol) was added in a dichloromethanesolution (80 mL) of(6-((tert-butoxycarbonylamino)methyl)benzothiazole-2-yl)methanol (1.65g, 5.61 mmol), and the resultant solution was stirred at roomtemperature for 16 hours. The reaction mixture was refined by silica gelcolumn chromatography, and the title compound was obtained as a whitesolid (1.43 g, 4.89 mmol).

¹H-NMR (400 MHz, CDCl₃) δ ppm: 10.16 (s, 1H), 8.20 (d, J=8.4 Hz, 1H),7.93 (d, J=1.6 Hz, 1H), 7.54 (dd, J=8.4 Hz, 1.6 Hz, 1H), 5.0 (br, 1H),4.50 (br d, J=6.0 Hz, 2H), 1.48 (s, 9H)

Step 7: Synthesis of2-((0-2-bromoethenyl)-6-((tert-butoxycarbonylamino)methyl)benzothiazole(19)

(Bromodifluormethyl) triphenylphosphonium bromide (2.70 g, 6.19 mmol)was suspended in THF (27.5 mL), and the resultant mixture was cooleddown to −78° C., added a THF solution (21 mL) of potassium tert-butoxide(703.5 mg, 6.27 mmol) at or below −55° C., and was stirred for 1 hour.Next, a THF solution (24.5 mL) of6-((tert-butoxycarbonylamino)methyl)benzothiazole-2-carboxaldehyde (1.43g, 4.89 mmol) was added, and the resultant mixture was stirred at −78°C. for 3.5 hours. After the reaction liquid was brought to near 0° C., asaturated sodium hydrogen carbonate aqueous solution (30 mL) was addedthereto, followed by water and ethyl acetate, and the resultant mixturewas separated. After the organic layer was dried and condensed underreduced pressure, refining was performed by silica gel columnchromatography, and the title compound was obtained as a white solid(0.64 g, 1.73 mmol).

¹H-NMR (400 MHz, CDCl₃) δ ppm: 7.95 (d, J=8.4 Hz, 1H), 7.77 (br s, 1H),7.40 (br d, J=8.4 Hz, 1H), 7.395 (d, J=14 Hz, 1H), 7.388 (d, J=14 Hz,1H), 4.9 (br, 1H), 4.43 (br d, J=6.0 Hz, 2H), 1.47 (s, 9H)

Step 8: Synthesis of(E)-5-(4-(6-((tert-butoxycarbonylamino)methyl)benzothiazole-2-yl)-3-butene-1-ynyl)-2-(N-(tert-butoxycarbonyl)-N-methyl)aminopyridine(20)

From 2-(N-(tert-butoxycarbonyl)-N-methylamino)-5-ethynylpyridine (0.83g, 3.57 mmol) and2-((E)-2-bromoetheny0-6-((tert-butoxycarbonylamino)methyl)benzothiazole(0.64 g, 1.73 mmol), the title compound was obtained (0.68 g, 1.31 mmol)in the same procedure as step 5 of following synthesis example 33.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 8.49 (d, J=1.6 Hz, 1H), 7.96 (d, J=8.4Hz, 1H), 7.81 (d, J=8.8 Hz, 1H), 7.78 (br s, 1H), 7.70 (dd, J=8.8 Hz,2.4 Hz, 1H), 7.40 (br d, J=8.4 Hz, 1H), 7.25 (d, J=16.0 Hz, 1H), 6.84(d, J=16.0 Hz, 1H), 4.95 (br, 1H), 4.45 (br d, J=5.2 Hz, 2H), 3.40 (s,3H), 1.54 (s, 9H), 1.48 (s, 9H)

Step 9: Synthesis of(E)-5-(4-(6-(aminomethyl)benzothiazole-2-yl)-3-butene-1-ynyl)-2-(methylamino)pyridine(PBB3.2N)

(E)-5-(4-(6-((tert-butoxycarbonylamino)methyl)benzothiazole-2-yl)-3-butene-1-ynyl)-2-(N-(tert-butoxycarbonyl)-N-methyl)aminopyridine(0.28 g, 0.54 mmol) was added to a liquid mixture of dichloromethane(4.4 mL) and trifluoroacetic acid (4.4 mL), and the resultant liquidmixture was stirred at room temperature for 3.5 hours, and, after that,condensed at reduced pressure. A saturated sodium hydrogen carbonateaqueous solution was added to the residue, and, after stirring for awhile, the solid was filtered, washed several times with water, anddried at reduced pressure at 25° C., and the title compound was obtainedas an orange powdered solid (168.5 mg, 0.527 mmol).

PBB3.2N: ¹H-NMR (400 MHz, CD₃OD) δ ppm: 8.13 (d, J=1.6 Hz, 1H), 7.93 (d,J=1.2 Hz, 1H), 7.89 (d, J=8.4 Hz, 1H), 7.52-7.48 (m, 2H), 7.16 (d,J=16.0 Hz, 1H), 6.94 (d, J=16.0 Hz, 1H), 6.50 (dd, J=8.8 Hz, 0.4 Hz,1H), 3.94 (s, 2H), 2.89 (s, 3H)

Synthesis Embodiment 12 Synthesis of2-((1E,3E)-4-(4-aminophenyl)buta-1,3-dienyl)-6-methoxybenzo[d]thiazole-5-ol(Core1-4)

Step 1: Synthesis of 1-(benzyloxy)-4-bromo-2-methoxybenzene (2)

K₂CO₃ (30.5 g, 221 mmol) and benzyl bromide (18.9 g, 171 mmol) wereadded in a DMF solution (150 mL) of 1 (15 g, 73.8 mmol), and theresultant solution was stirred at 100° C. for 2 hours. The reaction wasfinished by adding water, and the organic layer was extracted withEtOAc. The combined organic phase was condensed. The crude product wasrefined, and 2 (17.6 g, 86%) was obtained.

Step 2: Synthesis of 1-(benzyloxy)-4-bromo-2-methoxy-5-nitrobenzene (3)

2 (6.73 g, 24 mmol) was added in a glacial acetic acid solution (96 mL)of concentrated HNO₃ (20 mL, 418 mmol) at −10° C., and the resultantsolution was stirred for 2 hours. The suspended solid was filtered anddried, and 3 (7.6 g, 97%) was obtained.

Step 3: Synthesis of 5-(benzyloxy)-2-bromo-4-methoxyaniline (4)

3 (8 g, 23.7 mmol) was added in an ethanol (200 mL)-water (20 mL),solution, and concentrated HCl (5 mL) was added in the resultantsolution dropwise at 0° C. To this, metal powder (7.95 g, 142 mmol) wasadded at 0° C., and the resultant mixture was stirred for 2 hours atroom temperature. The reacting mass was filtered through a celite bed,the filtrate was basified with 10 N NaOH, and the organic layer wasextracted with EtOAc. The combined organic phase was condensed. Thecrude product was refined, and 4 (5.1 g, 70%) was obtained.

Step 4: Synthesis of N-(5-(benzyloxy)-2-bromo-4-methoxyphenyl)acetamide(5)

Acetic anhydride (1.56 mL, 16.56 mmol) was added in a pyridine solution(30 mL) of 4 (5.1 g, 16.56 mmol) at 0° C., and the resultant solutionwas stirred at room temperature for 1 hour. The reacting mass wascondensed under reduced pressure, the resulting residue was diluted withwater, and the organic layer was extracted with EtOAc. The combinedorganic phase was condensed. By refining the crude product, 5 (5.0 g,86%) was obtained.

Step 5: Synthesis of(N-(5-(benzyloxy)-2-bromo-4-methoxyphenyl)ethanethioamide (6)

Pyridine (2.5 mL, 28.5 mmol) and Lawesson's reagent (7.5 g, 18.6 mmol)were added in a stirred toluene solution (50 mL) of 5 (5.0 g, 14.3mmol), and the reaction mixture was stirred at 120° C. for 2 hours. Thereaction mixture was cooled down to room temperature, and the solventwas removed. After that, water was added, and the organic layer wasextracted with EtOAc. The combined organic phase was condensed. Thecrude product was refined, and 6 (3.2 g, 61%) was obtained.

Step 6: Synthesis of 5-(benzyloxy)-6-methoxy-2-methylbenz[d]thiazole (7)

NaH (0.286 g, 1.2 mmol) was added in an NMP solution (200 mL) of 6 (2.9g, 7.9 mmol) at room temperature. The reaction mixture was stirred at150° C. for 2 hours. After that, the reaction was cooled down to roomtemperature and quenched with ice water, and the organic layer wasextracted with EtOAc. The combined organic phase was condensed. Thecrude product was refined by column chromatography, and 7 (1.5 g, 66%)was obtained.

Step 7: Synthesis of 6-methoxy-2-methylbenz[d]thiazole-5-ol (8)

In a dichloromethane solution (35 mL) of 7 (0.92 g, 3.22 mmol) anddimethylaniline (2.49 g, 20.9 mmol), AlCl₃ (2.36 g, 17.7 mmol) was addedat −5° C. The reaction substance was stirred at −5° C. for 10 minutes,and, after that, quenched by adding ice water, and the organic layer wasextracted with dichloromethane. The combined organic phase wascondensed. The crude product was refined by column chromatography, and 8(0.52 g, 82%) was obtained.

Step 8: Synthesis of5-(tert-butyldimethylsilyloxy)-6-methoxy-2-methylbenz[d]thiazole (9)

Imidazole (0.583 g, 8.6 mmol) was added in a DMF solution (5 mL) of 8(0.52 g, 2.66 mmol) at 0° C. The reaction liquid was stirred at 0° C.for 10 minutes, and TBDMSCl (0.95 g, 6.3 mmol) was added. The reactionmixture was stirred for 2.5 hours at room temperature. The reaction wasfinished by adding water, and the organic layer was extracted withdichloromethane. The combined organic phase was condensed. The crudeproduct was refined by column chromatography, and 9 (0.55 g, 66%) wasobtained.

Step 9: Synthesis of2-(bromomethyl)-5-(tert-butyldimethylsilyloxy)-6-methoxybenzo[d]thiazole(10)

NBS (0.690 g, 3.88 mmol) and the catalyst quantity of AIBN were added ina CCl₄ solution (10 mL) of 9 (1 g, 3.23 mmol) at room temperature.Philips *IR 250 W* lamp was placed at a certain distance from thereaction flask so as to maintain the reflux. The reaction mixture wasrefluxed for 2 hours, and, after that, diluted with dichloromethane andwashed with water. The organic phase was separated and condensed. Thecrude product was refined by column chromatography, and 10 (0.55 g, 44%)was obtained.

Step 10: Synthesis of diethyl(5-hydroxy-6-methoxybenzo[d]thiazole-2-yl)methylphosphonate (11)

A mixture of 10 (0.55 g, 1.4 mmol) and triethyl phosphite (0.23 g, 1.4mmol) was heated to 100° C. for 2 hours. The crude product was refinedby column chromatography, and 11 (0.31 g, 65%) was obtained.

Step 11: Synthesis of6-methoxy-2((1E,3E)-4-(4-nitrophenyl)buta-1,3-dienyl)benz[d]thiazole-5-ol(12)

Sodium methoxide (0.1 g, 1.86 mmol) was added in a stirred DMF solution(3 mL) of 11 (0.33 g, 0.99 mmol) at 0° C., and the resultant solutionwas stirred for 30 minutes at the same temperature. (4-nitrophenyl)acrylic aldehyde (0.11 g, 0.62 mmol) was added to this, and theresultant solution was stirred for 30 minutes. The reaction was quenchedwith water, and acidified with citric acid. After that, the reactionmixture was extracted with EtOAc. The combined organic phase wascondensed to dryness, and 12 (210 mg) was obtained. Without refining,the step moved on to the next step.

Step 12: Synthesis of2-((1E,3E)-4-(4-aminophenyl)buta-1,3-dienyl)-6-methoxybenzo[d]thiazole-5-ol(Core1-4)

A EtOH liquid mixture (10 mL) of 12 (0.55 g, 1.6 mmol), iron powder(0.73 g, 12.8 mmol) and a saturated NH₄Cl solution (2 mL) was heated to80° C. for 1 hour. After that, the reacting mass was cooled, andfiltered through a celite bed. The filtrate was condensed, the resultingresidue was diluted with water, and the reaction mixture was extractedwith EtOAc. The organic phase was condensed to dryness, and 450 mg ofCore1-4 was obtained. 180 mg of that was applied to preparative HPLC,and Core1-4 (73 mg) was obtained.

Core1-4: ¹H NMR (400 MHz, DMSO-d₆) δ 7.55 (s, 1H), 7.39 (d, J=8.0 Hz,2H), 7.31-7.20 (m, 2H), 7.04-6.77 (m, 5H), 4.8 (bs, 1H) 3.94 (s, 3H).

Synthesis Embodiment 13 Synthesis ofN-(4-((1E,3E)-4-(5,6-dimethoxybenzo[d]thiazole-2-yl)buta-1,3-dienypphenyl)acetamide (Core1-5)

Core1-5 was synthesized according to the following synthesis scheme.

Step 1: Synthesis of (E)-3-(4-nitrophenyl)acrylic aldehyde (B)

To a liquid mixture of 4-nitrobenzaldehyde (25 g, 165 mmol) andacetaldehyde (50 mL, 900 mmol), a 20% potassium hydroxide MeOH solution(6 mL) was added dropwise at 0° C. to −5° C., until an alkaline reactionwas achieved. The reaction liquid was stirred at the same temperatureuntil the reaction mixture solidified. Acetic anhydride (80 mL) wasadded to this, and the mixture was heated for 30 minutes at 100° C.After that, the solution was poured in warm water (500 mL), andconcentrated HCl (32 mL) was added thereto. The resulting mixture washeated at 100° C. for 20 minutes. The resulting mixture was allowed tostand overnight, and the crystals were collected by filtering and washedwith water, and B (20 g, 68%) was obtained.

Step 2: Synthesis of 2-(bromomethyl)-5,6-dimethoxybenzo[d]thiazole (2)

NBS (5.11 g, 28.7 mmol) and the catalyst quantity of AIBN were added ina CCl₄ solution (50 mL) of 1 (5 g, 23.9 mmol) at room temperature.Philips *IR 250 W* lamp was placed at a certain distance from thereaction flask so as to maintain the reflux. The reaction mixture wasrefluxed for 2 hours, and, after that, diluted with dichloromethane, andwashed with water. The organic phase was separated and condensed. Thecrude product was refined, and 2 (3.0 g, 43%) was obtained.

Step 3: Synthesis of diethyl(5,6-dimethoxybenzo[d]thiazole-2-yl)methylphosphonate (3)

A mixture of 2 (3 g, 10.46 mmol) and triethyl phosphite (2 g, 11.45mmol) was heated to 100° C. for 2 hours. The crude product was refinedby column chromatography, and 3 (3.3 g, 92%) was obtained.

Step 4: Synthesis of5,6-dimethoxy-2-((1E,3E)-4-(4-nitrophenyl)buta-1,3-dienyl)benz[d]thiazole(4)

Sodium methoxide (0.085 g, 1.6 mmol) was added in a stirred DMF solution(3 mL) of 3 (0.30 g, 0.85 mmol) at 0° C., and the resultant solution wasstirred at the same temperature for 30 minutes. (4-nitrophenyl)acrylicaldehyde (0.14 g, 0.79 mmol) was added to this, and the resultantmixture was stirred for 30 minutes. The reaction was quenched withwater, and acidified with citric acid. After that, the reaction liquidmixture was extracted with EtOAc, the combined organic phase wascondensed, and refined by column chromatography, and 4 (0.21 g, 65%) wasobtained.

Step 5: Synthesis of4-((1E,3E)-4-(5,6-dimethoxybenzo[d]thiazole-2-yl)buta-1,3-dienyl)aniline(5)

Iron powder (0.06 g, 1.1 mmol) and a saturated ammonium chloride aqueoussolution (1 mL) were added in an EtOH solution (1 mL) of 4 (0.05 g, 0.13mmol). The reaction mixture was refluxed for 30 minutes. After that, thereacting mass was cooled and filtered through a celite bed. The filtratewas condensed to dryness, and 5 (40 mg, 88%) was obtained.

Step 6: Synthesis ofN-(4-((1E,3E)-4-(5,6-dimethoxybenzo[d]thiazole-2-yl)buta-1,3-dieneyl)phenyl)acetamide(Core1-5)

Triethylamine (0.037 g, 0.37 mmol) and acetic anhydride (0.029 g, 0.37mmol) were added in a dichloromethane solution (2 mL) of 5 (0.05 g, 0.15mmol). The reaction liquid mixture was stirred at room temperature for 1hour. The reaction liquid mixture was diluted with water and extractedwith dichloromethane. The combined organic phase was condensed andrefined by preparative HPLC, and Core1-5 (0.02 g, 36%) was obtained.

Core1-5: ¹H-NMR (400 MHz, choloroform-d) δ 7.60-6.74 (m, 10H), 3.98 (s,6H), 2.21 (s, 3H).

Synthesis Embodiment 14 Synthesis of3-(4-((1E,3E)-4-(5,6-dimethozybenzo[d]thiazole-2-yl)buta-1,3-dienyl)phenylamino)propan-1-ol(Core1-11)

Core1-11 was synthesized according to the following synthesis scheme.

Step 1: Synthesis of (E)-3-(4-nitrophenyl)acrylic Aldehyde (B)

A 20% potassium hydroxide MeOH solution (6 mL) was added dropwise to aliquid mixture of 4-nitrobenzaldehyde (25 g, 165 mmol) and acetaldehyde(50 mL, 900 mmol), at O° C. to −5° C., until an alkaline reaction wasachieved. The reaction liquid was stirred at the same temperature untilthe reaction mixture solidified. Acetic anhydride (80 mL) was added tothis, and the mixture was heated for 30 minutes at 100° C. After that,the solution was poured in warm water (500 mL), and concentrated HCl (32mL) was added thereto. The resulting mixture was heated at 100° C. for20 minutes. The resulting mixture was allowed to stand overnight, thecrystals were collected by filtering and washed with water, and B (20 g,68%) was obtained.

Step 2: Synthesis of 2-(bromomethyl)-5,6-dimethoxybenzo[d]thiazole (2)

NBS (5.11 g, 28.7 mmol) and the catalyst quantity of AIBN were added ina CCl₄ solution (50 mL) of 1 (5 g, 23.9 mmol) at room temperature.Philips *IR 250 W* lamp was placed at a certain distance from thereaction flask so as to maintain the reflux. The reaction mixture wasrefluxed for 2 hours, and, after that, diluted with dichloromethane andwashed with water. The organic phase was separated and condensed. Thecrude product was refined, and 2 (3.0 g, 43%) was obtained.

Step 3: Synthesis of diethyl(5,6-dimethoxybenzo[d]thiazole-2-yl)methylphosphonate (3)

A mixture of 2 (3 g, 10.46 mmol) and triethyl phosphite (2 g, 11.45mmol) was heated to 100° C. for 2 hours. The crude product was refinedby column chromatography, and 3 (3.3 g, 92%) was obtained.

Step 4: Synthesis of5,6-dimethoxy-2-((1E,3E)-4-(4-nitrophenyl)buta-1,3-dienyl)benz[d]thiazole(4)

Sodium methoxide (0.085 g, 1.6 mmol) was added in a stirred DMF solution(3 mL) of 3 (0.30 g, 0.85 mmol) at 0° C., and the resultant solution wasstirred at the same temperature for 30 minutes. To this,(4-nitrophenyl)acrylic aldehyde (0.14 g, 0.79 mmol) was added, and theresultant solution was stirred for 30 minutes. The reaction was quenchedwith water, and acidified with citric acid. After that, the reactionliquid mixture was extracted with EtOAc, the combined organic phase wascondensed, the crude product was refined by column chromatography, and 4(0.21 g, 65%) was obtained.

Step 5: Synthesis of4-((1E,3E)-4-(5,6-dimethoxybenzo[d]thiazole-2-yl)buta-1,3-dienyl)aniline(5)

Iron powder (0.06 g, 1.1 mmol) and a saturated ammonium chloride aqueoussolution (1 mL) were added in an EtOH solution (1 mL) of 4 (0.05 g, 0.13mmol). The reaction mixture was refluxed for 30 minutes. After that, thereacting mass was cooled, and filtered through celite bed. The filtratewas condensed to dryness, and 5 (40 mg, 88%) was obtained.

Step 6: Synthesis of3-(4-((1E,3E)-4-(5,6-dimethoxybenzo[d]thiazole-2-yl)buta-1,3-diene-1-yl)phenylamino)propan-1-ol(Core1-11)

Triethylamine (0.22 g, 2.21 mmol) and 3-bromo-1-propanol (0.3 g, 2.21mmol) were added in a dichloromethane solution (10 mL) of 5 (0.3 g, 0.88mmol). The reaction liquid mixture was stirred at room temperature for 1hour. The reaction liquid mixture was diluted with water, and extractedwith dichloromethane. The combined organic phase was condensed andrefined by preparative HPLC, and Core1-11 (0.06 g, 17%) was obtained.

Core1-11: ¹H NMR (400 MHz, DMSO-d₆) δ 7.59 (s, 1H), 7.45 (s, 1H),7.36-7.17 (m, 3H), 6.91-6.79 (m, 3H), 6.60 (d, J=8.3 Hz, 2H), 3.84 (d,J=2.0 Hz, 6H), 3.50 (t, J=6.2, 6.2 Hz, 2H), 3.11 (t, J=7.0, 7.0 Hz, 2H),1.70 (m, 2H).

Synthesis Embodiment 15 Synthesis of4-((1E,3E)-4-(5,6-dimethoxybenzo[d]thiazole-2-yl)buta-1,3-dienyl)-N-isopropylaniline(Core1-15)

Core1-15 was synthesized according to the following synthesis scheme:

Step 1: Synthesis of (E)-3-(4-nitrophenyl)acrylic aldehyde(B)

A 20% potassium hydroxide MeOH solution (6 mL) was added dropwise in aliquid mixture of 4-nitrobenzaldehyde (25 g, 165 mmol) and acetaldehyde(50 mL, 900 mmol), at O° C. to −5° C., until an alkaline reaction wasachieved. The reaction liquid was stirred at the same temperature untilthe reaction mixture solidified. Acetic anhydride (80 mL) was added tothis, and the mixture was heated for 30 minutes at 100° C. After that,the solution was poured in warm water (500 mL), and concentrated HCl (32mL) was added thereto. The resulting mixture was heated at 100° C. for20 minutes. The resulting mixture was allowed to stand overnight, thecrystals were collected by filtering and washed with water, and B (20 g,68%) was obtained.

Step 2: Synthesis of 2-(bromomethyl)-5,6-dimethoxybenzo[d]thiazole (2)

NBS (5.11 g, 28.7 mmol) and the catalyst quantity of AIBN were added ina CCl₄ solution (50 mL) of 1 (5 g, 23.9 mmol) at room temperature.Philips *IR 250 W* lamp was placed at a certain distance from thereaction flask so as to maintain the reflux. The reaction mixture wasrefluxed for 2 hours, and, after that, diluted with dichloromethane andwashed with water. The organic phase was separated and condensed. Thecrude product was refined, and 2 (3.0 g, 43%) was obtained.

Step 3: Synthesis ofdiethyl(5,6-dimethoxybenzo[d]thiazole-2-yl)methylphosphonate (3)

A mixture of 2 (3 g, 10.46 mmol) and triethyl phosphite (2 g, 11.45mmol) was heated to 100° C. for 2 hours. The crude product was refinedby column chromatography, and 3 (3.3 g, 92%) was obtained.

Step 4: Synthesis of5,6-dimethoxy-2-((1E,3E)-4-(4-nitrophenyl)buta-1,3-dienyl)benz[d]thiazole(4)

Sodium methoxide (0.085 g, 1.6 mmol) was added in a stirred DMF solution(3 mL) of 3 (0.30 g, 0.85 mmol) at O° C., and the resultant solution wasstirred at the same temperature for 30 minutes. (4-nitrophenyl)acrylicaldehyde (0.14 g, 0.79 mmol) was added to this, and the resultantmixture was stirred for 30 minutes. The reaction was quenched withwater, and acidified with citric acid. After that, the reaction liquidmixture was extracted with EtOAc, the combined organic phase wascondensed and refined by column chromatography, and 4 (0.21 g, 65%) wasobtained.

Step 5: Synthesis of4-((1E,3E)-4-(5,6-dimethoxybenzo[d]thiazole-2-yl)buta-1,3-dienyl)aniline(5)

Iron powder (0.06 g, 1.1 mmol) and a saturated ammonium chloride aqueoussolution (1 mL) were added in an EtOH solution (1 mL) of 4 (0.05 g, 0.13mmol). The reaction mixture was refluxed for 30 minutes. After that, thereacting mass was cooled, and filtered through a celite bed. Thefiltrate was condensed to dryness, and 5 (40 mg, 88%) was obtained.

Step 6: Synthesis of4-((1E,3E)-4-(5,6-dimethoxybenzo[d]thiazole-2-yl)buta-1,3-diene-1-yl)-N-isopropylaniline(Core1-15)

Triethylamine (0.037 g, 0.37 mmol) and 2-bromopropane (0.045 g, 0.37mmol) were added in a dichloromethane solution (2 mL) of 5 (0.05 g, 0.15mmol). The reaction liquid mixture was stirred at room temperature for 1hour. The reaction liquid mixture was diluted with water, and extractedwith dichloromethane. The combined organic phase was condensed andrefined by preparative HPLC, and Core1-15 (0.023 g, 41%) was obtained.

Core1-15: ¹H NMR (400 MHz, DMSO-d₆) δ 7.72-7.17 (m, 7H), 7.10-6.55 (m,6H), 5.76 (s, 1H), 3.84 (s, 6H), 1.23 (m, 1H) 1.16 (dd, J=6.1, 3.3 Hz,6H).

Synthesis Embodiment 16 Synthesis of4-((1E,3E)-4-(5,6-dimethoxybenzo[d]thiazole-2-yl)buta-1,3-dienyl)-N-(hepta-1,6-diene-4-yl)aniline(Core1-20)

Core1-20 was synthesized according to the following synthesis scheme:

Step 1: Synthesis of (E)-3-(4-nitrophenyl)acrylic aldehyde (B)

A 20% potassium hydroxide MeOH solution (6 mL) was added dropwise in aliquid mixture of 4-nitrobenzaldehyde (25 g, 165 mmol) and acetaldehyde(50 mL, 900 mmol), at 0° C. to −5° C., until an alkaline reaction wasachieved. The reaction liquid was stirred at the same temperature untilthe reaction mixture solidified. Acetic anhydride (80 mL) was added tothis, and the mixture was heated for 30 minutes at 100° C. After that,the solution was poured in warm water (500 mL), and concentrated HCl (32mL) was added. The resulting mixture was heated at 100° C. for 20minutes. The resulting mixture was allowed to stand overnight, thecrystals were collected by filtering and washed with water, and B (20 g,68%) was obtained.

Step 2: Synthesis of 2-(bromomethyl)-5,6-dimethoxybenzo[d]thiazole (2)

NBS (5.11 g, 28.7 mmol) and the catalyst quantity of AIBN were added ina CCl₄ solution (50 mL) of 1 (5 g, 23.9 mmol) at room temperature. APhilips *IR 250 W* lamp was placed at a certain distance from thereaction flask so as to maintain the reflux. The reaction mixture wasrefluxed for 2 hours, and, after that, diluted with dichloromethane andwashed with water. The organic phase was separated and condensed. Thecrude product was refined, and 2 (3.0 g, 43%) was obtained.

Step 3: Synthesis of diethyl(5,6-dimethoxybenzo[d]thiazole-2-yl)methylphosphonate (3)

A mixture of 2 (3 g, 10.46 mmol) and triethyl phosphite (2 g, 11.45mmol) was heated to 100° C. for 2 hours. The crude product was refinedby column chromatography, and 3 (3.3 g, 92%) was obtained.

Step 4: Synthesis of5,6-dimethoxy-2-((1E,3E)-4-(4-nitrophenyl)buta-1,3-dienyl)benz[d]thiazole(4)

Sodium methoxide (0.085 g, 1.6 mmol) was added in a stirred DMF solution(3 mL) of 3 (0.30 g, 0.85 mmol) at 0° C., and the resultant solution wasstirred at the same temperature for 30 minutes. (4-nitrophenyl)acrylicaldehyde (0.14 g, 0.79 mmol) was added to this, and the resultingsolution was stirred for 30 minutes. The reaction was quenched withwater, and acidified with citric acid. After that, the reaction liquidmixture was extracted with EtOAc, the combined organic phase wascondensed and refined by column chromatography, and 4 (0.21 g, 65%) wasobtained.

Step 5: Synthesis of4-((1E,3E)-4-(5,6-dimethoxybenzo[d]thiazole-2-yl)buta-1,3-dienyl)aniline(5)

Iron powder (0.06 g, 1.1 mmol) and a saturated ammonium chloride aqueoussolution (1 mL) were added in an EtOH solution (1 mL) of 4 (0.05 g, 0.13mmol). The reaction mixture was refluxed for 30 minutes. After that, thereacting mass was cooled and filtered through a celite bed. The filtratewas condensed to dryness, and 5 (40 mg, 88%) was obtained.

Step 6: Synthesis of4-((1E,3E)-4-(5,6-dimethoxybenzo[d]thiazole-2-yl)buta-1,3-diene-1-yl)-N-(hepta-1,6-diene-4-yl)aniline(Core1-20)

Triethylamine (0.037 g, 0.37 mmol) and allyl bromide (0.044 g, 0.37mmol) were added in a dichloromethane solution (2 mL) of 5 (0.05 g, 0.15mmol). The reaction liquid mixture was stirred at room temperature for 1hour. The reaction liquid mixture was diluted with water and extractedwith dichloromethane. The combined organic phase was condensed andrefined by preparative HPLC, and Core1-20 (0.026 g, 41.2%) was obtained.

Core1-20 ¹H NMR (400 MHz, DMSO-d₆) δ 7.59-7.18 (m, 5H), 6.93-6.81 (m,3H), 6.68 (d, J=8.6 Hz, 2H), 5.86 (m, 2H), 5.24-5.06 (m, 4H), 3.97 (d,J=5.2 Hz, 4H), 3.84 (d, J-2.1 Hz, 6H).

Synthesis Embodiment 17 Synthesis ofN-(5-((1E,3E)-4-(5,6-dimethozybenzo[d]thiazole-2-yl)buta-1,3-dienyl)pyridine-2-yl)acetamide(Core2-9)

Core2-9 was synthesized according to the following synthesis scheme:

Step 1: Synthesis of (E)-3-(6-bromopyridine-3-yl)acrylic aldehyde(B)

In a THF solution (5 mL) of 2,5-dibromopyridine (2.37 g, 10 mmol),2-propylmagnesiumchloride (in THF, 2.0 M, 5 mL, 10 mmol) was added atroom temperature. The resulting suspension was stirred for 1 hour, and,after that, cooled down to 0° C. 3-dimethylaminoacrolein (1.3 mL, 12.36mmol) was added, and the mixture was warmed to room temperature andstirred for 2 hours. The reaction was finished by adding ice at 0° C.,and acidified with 2N HCl. After that, the resultant mixture was dilutedwith EtOAc and washed with water. The organic phase was separated andcondensed. The crude product was refined, and B (0.45 g, 21%) wasobtained.

Step 2: Synthesis of 2-(bromomethyl)-5,6-dimethoxybenzo[d]thiazole (2)

NBS (5.11 g, 28.7 mmol) and the catalyst quantity of AIBN were added ina CCl₄ solution (50 mL) of 1 (5 g, 23.9 mmol) at room temperature.Philips *IR 250 W* lamp was placed at a certain distance from thereaction flask so as to maintain the reflux. The reaction mixture wasrefluxed for 2 hours, and, after that, diluted with dichloromethane, andwashed with water. The organic phase was separated and condensed. Thecrude product was refined, and 2 (3.0 g, 43%) was obtained.

Step 3: Synthesis of diethyl(5,6-dimethoxybenzo[d]thiazole-2-yl)methylphosphonate (3)

A mixture of 2 (3 g, 10.46 mmol) and triethyl phosphite (2 g, 11.45mmol) was heated to 100° C. for 2 hours. The crude product was refinedby column chromatography, and 3 (3.3 g, 92%) was obtained.

Step 4: Synthesis of2-((1E,3E)-4-(6-bromopyridine-3-yl)buta-1,3-dienyl)-5,6-dimethoxybenzo[d]thiazole(4)

Sodium methoxide (0.10 g, 1.96 mmol) was added in a stirred DMF solution(5 mL) of 3 (0.50 g, 1.44 mmol) at 0° C., and was stirred at the sametemperature for 30 minutes. B (0.27 g, 1.3 mmol) was added to this, andthe resultant mixture was stirred for 30 minutes, and the reaction wasquenched with water and acidified with citric acid. After that, thereaction mixture was extracted with EtOAc, the combined organic phasewas condensed and refined by column chromatography, and 4 (0.512 g, 85%)was obtained.

Step 5: Synthesis of5-((1E,3E)-4-(5,6-dimethoxybenzo[d]thiazole-2-yl)buta-1,3-diene-1-yl)pyridine-2-amine(5)

A mixture of 4 (0.5 g, 1.24 mmol) and ammonia water (10 mL) was put in asealed tube, and the reaction mixture was refluxed for 4 hours. Thereaction mixture was condensed and refined by column chromatography, and5 (0.2 g, 47.6%) was obtained.

Step 6: Synthesis ofN-(5-((1E,3E)-4-(5,6-dimethoxybenzo[d]thiazole-2-yl)buta-1,3-diene-1-yl)pyridine-2-yl)acetamide(Core2-9)

Triethylamine (0.148 g, 1.47 mmol) and acetic anhydride (0.15 g, 1.47mmol) were added in a dichloromethane solution (10 mL) of 5 (0.2 g,0.589 mmol). The reaction liquid mixture was stirred at room temperaturefor 1 hour. The reaction liquid mixture was diluted with water andextracted with dichloromethane. The combined organic phase was condensedand refined by preparative HPLC, and Core2-9 (0.04 g, 18%) was obtained.

Core2-9: ¹H NMR (400 MHz, DMSO-d₆) δ 10.62 (s, 1H), 8.45 (s, 1H), 8.09(d, J=8.7 Hz, 1H), 8.00 (dd, J=8.7, 2.3 Hz, 1H), 7.63 (s, 1H), 7.49 (s,1H), 7.38-7.16 (m, 2H), 6.98 (m, 2H), 3.85 (s, 6H), 2.10 (s, 3H).

Synthesis Embodiment 18 Synthesis of3-(5-((1E,3E)-4-(5,6-dimethoxybenzo[d]thiazole-2-yl)buta-1,3-dienyl)pyridine-2-ylamino)propan-1-ol(Core2-10)

Core2-10 was synthesized according to the following synthesis scheme:

Step 1: Synthesis of (E)-3-(6-bromopyridine-3-yl)acrylic aldehyde (B)

In a THF solution (5 mL) of 2,5-dibromopyridine (2.37 g, 10 mmol),2-propylmagnesiumchloride (in THF, 2.0 M, 5 mL, 10 mmol) was added atroom temperature. The resulting suspension was stirred for 1 hour, and,after that, cooled down to 0° C. 3-dimethylaminoacrolein (1.3 mL, 12.36mmol) was added, and the mixture was warmed to room temperature andstirred for 2 hours. The reaction was finished by adding ice at 0° C.,and acidified with 2N HCl. After that, the resultant mixture was dilutedwith EtOAc and washed with water. The organic phase was separated andcondensed. The crude product was refined, and B (0.45 g, 21%) wasobtained.

Step 2: Synthesis of 2-(bromomethyl)-5,6-dimethoxybenzo[d]thiazole (2)

NBS (5.11 g, 28.7 mmol) and the catalyst quantity of AIBN were added ina CCl₄ solution (50 mL) of 1 (5 g, 23.9 mmol) at room temperature.Philips *IR 250 W* lamp was placed at a certain distance from thereaction flask so as to maintain the reflux. The reaction mixture wasrefluxed for 2 hours, and, after that, diluted with dichloromethane, andwashed with water. The organic phase was separated and condensed. Thecrude product was refined, and 2 (3.0 g, 43%) was obtained.

Step 3: Synthesis ofdiethyl(5,6-dimethoxybenzo[d]thiazole-2-yl)methylphosphonate (3)

A mixture of 2 (3 g, 10.46 mmol) and triethyl phosphite (2 g, 11.45mmol) was heated to 100° C. for 2 hours. The crude product was refinedby column chromatography, and 3 (3.3 g, 92%) was obtained.

Step 4: Synthesis of2-((1E,3E)-4-(6-bromopyridine-3-yl)buta-1,3-dienyl)-5,6-dimethoxybenzo[d]thiazole(4)

Sodium methoxide (0.10 g, 1.96 mmol) was added in a stirred DMF solution(5 mL) of 3 (0.50 g, 1.44 mmol) at 0° C., and the resultant solution wasstirred at the same temperature for 30 minutes. B (0.27 g, 1.3 mmol) wasadded to this, the resultant solution was stirred for 30 minutes, andthe reaction was quenched with water and acidified with citric acid.After that, the reaction mixture was extracted with EtOAc, the combinedorganic phase was condensed and refined by column chromatography, and 4(0.512 g, 85%) was obtained.

Step 5: Synthesis of3-(5-((1E,3E)-4-(5,6-dimethoxybenzo[d]thiazole-2-yl)buta-1,3-dienyl)pyridin-2-ylamino)propan-1-ol(Core2-10)

In a sealed tube, a DMF liquid mixture (5 mL) of 4 (0.2 g, 0.49 mmol),3-aminopropanol (0.3 g, 4.96 mmol) and triethylamine (0.25 g, 2.48 mmol)was stirred, at 120° C., for 16 hours. The reaction mixture was dilutedwith water, refined by preparative HPLC, and Core2-10 (0.04 g, 20%) wasobtained.

Core2-10: ¹H NMR (400 MHz, chloroform-d) δ 9.87 (s, 1H), 8.03 (d, J=9.7Hz, 1H), 7.81 (s, 1H), 7.56 (s, 1H), 7.23 (d, J=13.9 Hz, 2H), 7.09 (d,J=15.4 Hz, 1H), 6.90 (m, 2H), 6.66 (d, J=15.5 Hz, 1H), 3.99 (d, J=2.5Hz, 6H), 3.82 (t, J=5.8, 5.8 Hz, 2H), 3.53 (t, J=6.7, 6.7 Hz, 2H), 1.97(m, 2H).

Synthesis Embodiment 19 Synthesis ofN,N-diallyl-5-((1E,3E)-4-(5,6-dimethoxybenzo[d]thiazole-2-yl)buta-1,3-dienyl)pyridine-2-amine(Core2-14)

Core2-14 was synthesized according to the following synthesis scheme:

Step 1: Synthesis of (E)-3-(6-bromopyridine-3-yl)acrylic aldehyde (B)

In a THF solution (5 mL) of 2,5-dibromopyridine (2.37 g, 10 mmol),2-propylmagnesiumchloride/chloride (in THF, 2.0 M, 5 mL, 10 mmol) wasadded at room temperature. The resulting suspension was stirred for 1hour, and, after that, cooled down to 0° C. 3-dimethylaminoacrolein (1.3mL, 12.36 mmol) was added, and the mixture was warmed to roomtemperature and stirred for 2 hours. The reaction was finished by addingice at 0° C., and acidified with 2N HCl. After that, the resultantmixture was diluted with EtOAc, and washed with water. The organic phasewas separated and condensed. The crude product was refined, and B (0.45g, 21%) was obtained.

Step 2: Synthesis of 2-(bromomethyl)-5,6-dimethoxybenzo[d]thiazole (2)

NBS (5.11 g, 28.7 mmol) and the catalyst quantity of AIBN were added ina CCl₄ solution (50 mL) of 1 (5 g, 23.9 mmol) at room temperature.Philips *IR 250 W* lamp was placed at a certain distance from thereaction flask so as to maintain the reflux. The reaction mixture wasrefluxed for 2 hours, and, after that, diluted with dichloromethane andwashed with water. The organic phase was separated and condensed. Thecrude product was refined, and 2 (3.0 g, 43%) was obtained.

Step 3: Synthesis of diethyl(5,6-dimethoxybenzo[d]thiazole-2-yl)methylphosphonate (3)

A mixture of 2 (3 g, 10.46 mmol) and triethyl phosphite (2 g, 11.45mmol) was heated to 100° C. for 2 hours. The crude product was refinedby column chromatography, and 3 (3.3 g, 92%) was obtained.

Step 4: Synthesis of2-((1E,3E)-4-(6-bromopyridine-3-yl)buta-1,3-dienyl)-5,6-dimethoxybenzo[d]thiazole(4)

Sodium methoxide (0.10 g, 1.96 mmol) was added in a stirred DMF solution(5 mL) of 3 (0.50 g, 1.44 mmol) at 0° C., and the resultant solution wasstirred at the same temperature for 30 minutes. B (0.27 g, 1.3 mmol) wasadded to this, and the resultant mixture was stirred for 30 minutes, andthe reaction was quenched with water and acidified with citric acid.After that, the reaction mixture was extracted with EtOAc, the combinedorganic phase was condensed and refined by column chromatography, and 4(0.512 g, 85%) was obtained.

Step 5: Synthesis of5-((1E,3E)-4-(5,6-dimethoxybenzo[d]thiazole-2-yl)buta-1,3-diene-1-yl)pyridine-2-amine(5)

A mixture of 4 (0.5 g, 1.24 mmol) and ammonia water (10 mL) was put in asealed tube, and the reaction mixture was refluxed for 4 hours. Thereaction mixture was condensed and refined by column chromatography, and5 (0.2 g, 47.6%) was obtained.

Step 6: Synthesis ofN,N-diallyl-5-((1E,3E)-4-(5,6-dimethoxybenzo[d]thiazole-2-yl)buta-1,3-diene-1-yl)pyridine-2-amine(Core2-14)

Triethylamine (0.148 g, 1.47 mmol) and allyl bromide (0.18 g, 1.47 mmol)were added in a dichloromethane solution (10 mL) of 5 (0.2 g, 0.589mmol). The reaction liquid mixture was stirred at room temperature for 1hour. The reaction liquid mixture was diluted with water and extractedwith dichloromethane. The combined organic phase was condensed andrefined by preparative HPLC, and Core2-14 (0.03 g, 12%) was obtained.

Core2-14: ¹H NMR (400 MHz, DMSO-d₆) δ 8.16 (d, J=2.5 Hz, 1H), 7.93-7.85(m, 1H), 7.61 (s, 1H), 7.47 (s, 1H), 7.26 (dd, J=15.3, 10.6 Hz, 1H),7.04 (dd, J=15.5, 10.6 Hz, 1H), 6.95-6.69 (m, 3H), 5.86 (m, 2H),5.21-5.13 (m, 4H), 4.16 (d, J=5.2 Hz, 4H), 3.84 (d, J=1.8 Hz, 6H).

Synthesis Embodiment 20-1 Synthesis of1-fluoro-2-(2-((1E,3E)-4-(6-(methylamino)pyridine-3-yl)buta-1,3-dienyl)benz[d]thiazole-6-yloxy)-2-hydroxymethyl-ethane(F0-PBB3 Analog)

An F0-PBB3 analog was synthesized according to the following synthesisscheme:

Step 1: Synthesis of Compound (30)

Under an argon atmosphere, tetrabutylammoniumfluorid (1.0Mtetrahydrofuran solution, 3.15 mL, 3.15 mmol) was added to the compound(23) (819 mg, 2.86 mmol), and the resultant mixture was heated toreflux. The reaction liquid was cooled down to room temperature, addedwater, and extracted with diethyl ether. After the organic layer waswashed with water and dried with anhydrous sodium sulphate, diethylether was distillated under reduced pressure. Methanol (4.3 mL) wasadded to the residue and the resultant mixture was cooled with ice, and,after 4N hydrochloric acid/dioxane (1.4 mL) was added thereto and thetemperature was raised to room temperature, the resultant mixture wasstirred all night. The reaction liquid was distillated under reducedpressure, tetrahydrofuran (4.0 mL) and imidazole (131 mg, 1.92 mmol)were added in reaction liquid, and the resultant liquid was cooled withice. After t-butyldimethylchlorosilane (247 mg, 1.64 mmol) was added inthe reaction liquid and the reaction liquid was heated to roomtemperature, the reaction liquid was stirred all night. The reactionliquid was added water and extracted with ethyl acetate. After theorganic layer was washed with water and saturated saline water and driedwith anhydrous sodium sulphate, the solvent was distillated underreduced pressure. By refining the residue by column chromatography(developing solvent: heptane/ethyl acetate=20/1→10/1), 199 mg of thetitle compound (30) was obtained.

Step 2: Synthesis of Compound (31)

Under an argon atmosphere, the compound (30) (180 mg, 0.86 mmol) andtriphenylphosphine (226 mg, 0.86 mmol) were added in a tetrahydrofuransolution (4.3 mL) of the compound (28) (140 mg, 0.43 mmol), and theresultant solution was cooled with ice. Diisopropyl azodicarboxylate(174 mg, 0.86 mmol) was added dropwise to the reaction liquid. Thereaction liquid was heated to room temperature, and, after having beenstirred all night, the reaction liquid was distillated under reducedpressure. By refining the residue by column chromatography (developingsolvent: heptane/ethyl acetate=5/1→1/1), 200 mg of the title compound(31) was obtained.

Step 3: Synthesis of Compound (6)

4N hydrochloric acid/dioxane (1.9 mL) was added in a tetrahydrofuransolution (5.7 mL) of the compound (31) (196 mg, 0.38 mmol), and theresultant solution was stirred. After the disappearance of the rawmaterial, the reaction liquid was cooled with ice, and, after havingbeen neutralized with a sodium hydrogen carbonate aqueous solution, thereaction liquid was extracted with ethyl acetate. After the organiclayer was washed with water and saturated saline water and dried withanhydrous sodium sulphate, the solvent was distillated under reducedpressure. By refining the residue by column chromatography (developingsolvent: heptane/ethyl acetate=2/1→1/4), 117 mg of the title compound(6) was obtained.

Compound (6): ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 8.20 (d, J=2.29 Hz, 1H),7.80 (dd, J=9.16 Hz, 1.83 Hz, 2H), 7.72 (d, J=2.29 Hz, 1H), 7.30 (dd,J=15.57 Hz, 10.08 Hz, 1H), 7.14 (dd, J=8.70 Hz, 2.29 Hz, 1H), 7.01 (dd,J=15.11 Hz, 10.53 Hz, 1H), 6.91 (d, J=15.57 Hz, 1H), 6.88 (d, J=15.57Hz, 1H), 6.70 (d, J=9.16 Hz, 1H), 5.07 (t, J=5.50 Hz, 1H), 4.55-4.85 (m,3H), 3.63-3.68 (m, 2H), 3.07 (s, 6H).

Synthesis Embodiment 20-2 Synthesis of1-fluoro-3-(2-((1E,3E)-4-(6-(methylamino)pyridine-3-yl)buta-1,3-dienyl)benz[d]thiazole-6-yloxy)propan-2-ol(FO-PBB3)

This can be synthesized by the same method as that of synthesis example20-1 above.

Synthesis Embodiment 21 Synthesis of(E)-1-fluoro-3-(2-(4-(6-(methylamino)pyridine-3-yl)buta-1-en-3-ynyl)benz[d]thiazole-6-yloxy)propan-2-ol(F0-PBB3.2)

This can be synthesized by the same method as that of synthesis example20-1 above.

Synthesis Embodiment 22 Synthesis of2-((1E,3E)-4-(2-fluoro-6-(methylamino)pyridine-3-yl)buta-1,3-dienyl)benz[d]thiazole-6-ol(F1-PBB3)

This can be synthesized by a similar method to that of synthesis example20-1 above.

Synthesis Embodiment 23 Synthesis of(E)-2-(4-(2-fluoro-6-(methylamino)pyridine-3-yl)buta-1-en-3-ynyl)benz[d]thiazole-6-ol(F1-PBB3.2)

This can be synthesized by a similar method to that of synthesis example20-1 above.

Synthesis Embodiment 24 Synthesis of2-((1E,3E)-4-(2-fluoro-6-(methylamino)pyridine-3-yl)buta-1,3-dienyl)benzofuran-5-ol(F1-PBBf3)

This can be synthesized by a similar method to that of synthesis example20-1 above.

Synthesis Embodiment 25 Synthesis of(E)-2-(4-(2-fluoro-6-(methylamino)pyridine-3-yl)buta-1-en-3-ynyl)benzofuran-5-ol(F1-PBBf3.2)

This can be synthesized by a similar method to that of synthesis example20-1 above.

Synthesis Embodiment 26 Synthesis of2-((1E,3E)-4-(6-(dimethylamino)pyridine-3-yl)buta-1,3-dienyl)quinoline-6-ol(PBQ3.0)

PBQ3.0 was synthesized according to the following synthesis scheme:

Step 1: Synthesis of Compound (21)

Under an argon atmosphere, after a tetrahydrofuran solution (80 mL) ofthe compound (18) (1213 mg, 4.00 mmol) was cooled with ice, sodiumhydride (60% oil, 960 mg, 24.00 mmol) was added. After the reactionliquid was heated to room temperature and stirred for 30 minutes, methyliodide (3407 mg, 24.00 mmol) was added. The reaction liquid was added inwater and stirred, and extracted with chloroform. After the organiclayer was washed with saturated saline water and dried with anhydroussodium sulphate, the solvent was distillated under reduced pressure. Byrefining the residue by column chromatography (developing solvent:chloroform→chloroform/methanol=97/3), 804 mg of the title compound (21)was obtained.

Step 2: Synthesis of PBQ3.0

Under an argon atmosphere, after a dichloromethane solution (80 mL) ofthe compound (21) (800 mg, 2.41 mmol) was cooled down to −40° C., borontribromide (1.0 M dichloromethane solution, 12.1 mL, 12.10 mmol) wasadded dropwise. The reaction liquid was heated to 5° C., and stirred allnight. After the reaction liquid was neutralized by adding a sodiumhydroxide aqueous solution under ice cold conditions, the organic layerwas extracted with chloroform. After the organic layer was washed withwater and saturated saline water and dried with anhydrous sodiumsulphate, the solvent was distillated under reduced pressure. Theresidue was refined by column chromatography (developing solvent:chloroform→chloroform/methanol=19/1). Methanol was added to the refinedproduct, the refined product was suspended and washed, and theprecipitate was filtered. The cake was dried under reduced pressure, and110 mg of PBQ3.0 was obtained.

PBQ3.0: ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 9.96 (s, 1H), 8.19 (d, J=2.29Hz, 1H), 8.05 (d, J=8.69 Hz, 1H), 7.79 (dd, J=9.15 Hz, 2.29 Hz, 1H),7.77 (d, J=9.15 Hz, 1H), 7.62 (d, J=8.69 Hz, 1H), 7.47 (dd, J=15.10 Hz,10.52 Hz, 1H), 7.26 (dd, J=9.15 Hz, 2.75 Hz, 1H), 7.09 (d, J=2.29 Hz,1H), 6.99 (dd, J=15.10 Hz, 10.52 Hz, 1H), 6.78 (d, J=15.55 Hz, 1H), 6.77(d, J=15.10 Hz, 1H), 6.68 (d, J=8.69 Hz, 1H), 3.06 (s, 6H).

Synthesis Embodiment 27 Synthesis of2-((1E,3E)-4-(6-(methylamino)pyridine-3-yl)buta-1,3-dienyl)quinoline-6-ol(PBQ3)

PBQ3 was synthesized according to the following synthesis scheme:

Step 1: Synthesis of Compound (17)

Under an argon atmosphere, after a tetrahydrofuran solution (200 mL) ofthe compound (8) (17.60 g, 56.9 mmol) was cooled with ice,tert-butyllithium (1.61M hexane solution, 38.9 mL, 62.6 mmol) was addeddropwise. After the reaction liquid was stirred for 60 minutes, atetrahydrofuran solution (100 mL) of the compound (16) (10.14 g, 56.9mmol) was added dropwise. The reaction liquid was heated to roomtemperature, and, after the disappearance of the raw material, thereaction liquid was added water, and extracted with chloroform. Afterthe organic layer was washed with water and saturated saline water anddried with anhydrous sodium sulphate, the solvent was distillated underreduced pressure. By refining the residue by column chromatography(developing solvent: chloroform→chloroform/ethyl acetate=19/1), 5.60 gof the title compound (17) was obtained.

Step 2: Synthesis of Compound (18)

Acetic acid (250 mL), iron (3.94 g, 70.5 mmol) and 12N hydrochloric acid(21 mL) were added in an ethanol solution (500 mL) of the compound (17)(5.00 g, 15.00 mmol). The reaction liquid was heated to 70° C., and,after the disappearance of the raw material was confirmed, cooled withice. After a sodium hydroxide aqueous solution was added dropwise to thereaction liquid and chloroform was added thereto, the reaction liquidwas filtered through celite. The filtrate was extracted with chloroform,and the organic layer was distillated under reduced pressure. Byrefining the residue by column chromatography (developing solvent:chloroform→chloroform/methanol=50/1), 3.01 g of the title compound (18)was obtained.

Step 3: Synthesis of Compound (19)

t-butyl alcohol (200 mL) and di-tert-butyl dicarbonate (1109 mg, 5.08mmol) were added in a tetrahydrofuran solution (40 mL) of the compound(18) (1402 mg, 4.62 mmol), and the resultant solution was heated to 35°C. and stirred all night. By distillating the reaction liquid underreduced pressure and refining the residue by column chromatography(developing solvent: chloroform→chloroform/methanol=24/1), 1078 mg ofthe title compound (19) was obtained.

Step 4: Synthesis of Compound (20)

Under an argon atmosphere, a tetrahydrofuran solution (133 mL) of thecompound (19) (1074 mg, 2.66 mmol) was cooled with ice, and sodiumhydride (60% oil, 319 mg, 7.99 mmol) was added thereto. After thereaction liquid was heated to room temperature and stirred for 30minutes, methyl iodide (1133 mg, 7.99 mmol) was added. The reactionliquid was added in water and stirred, and was extracted withchloroform. After the organic layer was washed with saturated salinewater and dried with anhydrous sodium sulphate, the solvent wasdistillated under reduced pressure. By refining the residue by columnchromatography (developing solvent:chloroform→chloroform/methanol=97/3), 701 mg of the title compound (20)was obtained.

Step 5: Synthesis of PBQ3

Under an argon atmosphere, after a dichloromethane solution (60 mL) ofthe compound (20) (670 mg, 1.60 mmol) was cooled down to −40° C. borontribromide (1.0 M dichloromethane solution, 8.02 mL, 8.02 mmol) wasadded dropwise. The reaction liquid was heated to 0° C. and stirred allnight. The reaction liquid was heated to 10° C. and stirred for 60minutes. After the reaction liquid was neutralized by adding methanoland sodium hydrogen carbonate under ice cold conditions, the organiclayer was extracted with dichloromethane. After the organic layer waswashed with water and saturated saline water and dried with anhydroussodium sulphate, the solvent was distillated under reduced pressure. Theresidue was refined by column chromatography (developing solvent:chloroform/methanol=99/1→9/1). Methanol was added to the refinedproduct, the refined product was suspended and washed, and theprecipitate was filtered. By drying the cake under reduced pressure, 120mg of PBQ3 was obtained.

PBQ3: ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 9.95 (s, 1H), 8.08 (d, J=2.29 Hz,1H), 8.04 (d, J=8.69 Hz, 1H), 7.77 (d, J=9.15 Hz, 1H), 7.69 (dd, J=8.69Hz, 2.29 Hz, 1H), 7.62 (d, J=8.69 Hz, 1H), 7.46 (dd, J=15.56 Hz, 10.98Hz, 1H), 7.26 (dd, J=9.15 Hz, 2.75 Hz, 1H), 7.08 (d, J=2.75 Hz, 1H),6.92 (dd, J=15.56 Hz, 10.98 Hz, 1H), 6.81 (q, J=5.03 Hz, 1H), 6.75 (d,J=15.55 Hz, 1H), 6.74 (d, J=15.10 Hz, 1H), 6.47 (d, J=9.15 Hz, 1H), 2.80(d, J=5.03 Hz, 3H).

Synthesis Embodiment 28 Synthesis of(E)-2-(4-(6-(dimethylamino)pyridine-3-yl)buta-1-en-3-ynyl)quinoline-6-ol(PBQ3.1)

PBQ3.1 was synthesized according to the following synthesis scheme:

Step 1: Synthesis of Compound (12)

Under an argon atmosphere, after a N,N-dimethylformamide solution (20mL) of 5-iodo-2-aminopyridine (11) (2200 mg, 10.0 mmol) was cooled withice, sodium hydride (60% oil, 1200 mg, 30.0 mmol) was added thereto. Thereaction liquid was heated to room temperature, and stirred for 30minutes. The reaction liquid was cooled with ice, and, after methyliodide (4258 mg, 30.0 mmol) was added thereto, the reaction liquid washeated to room temperature. After the disappearance of the raw material,the reaction liquid was added in water and stirred, and the organiclayer was extracted with ethyl acetate. After the organic layer waswashed with water and saturated saline water and dried with anhydroussodium sulphate, the solvent was distillated under reduced pressure. Byrefining the residue by column chromatography (developing solvent:heptane/ethyl acetate=99/1→24/1), 2086 mg of the title compound (12) wasobtained.

Step 2: Synthesis of Compound (13)

Under an argon atmosphere, copper iodide (191 mg, 1.00 mmol),2-propyn-1-ol (939 mg, 16.75 mmol) and dichlorobis (triphenylphosphine)palladium (II) (118 mg, 0.17 mmol) were added in a triethylaminesolution (8.17 mL, 58.61 mmol) of the compound (12) (2077 mg, 8.37mmol), and the resultant solution was stirred. After the disappearanceof the raw material was confirmed, the reaction liquid was filtered, andthe solvent was distillated under reduced pressure. By refining theresidue by column chromatography (developing solvent: heptane/ethylacetate=19/1→1/1), 1340 mg of the title compound (13) was obtained.

Step 3: Synthesis of Compound (14)

Under an argon atmosphere, triethylamine (2534 mg, 25.04 mmol) and apyridine sulfur trioxide complex (3623 mg, 22.76 mmol) were added in adimethylsulfoxide solution (37.9 mL) of the compound (13) (1337 mg, 7.59mmol), and the resultant solution was stirred. After the disappearanceof the raw material was confirmed, water was added in the reactionliquid, and the organic layer was extracted using ethyl acetate. Afterthe organic layer was washed with water and saturated saline water anddried with anhydrous sodium sulphate, the solvent was distillated underreduced pressure. By refining the residue by column chromatography(developing solvent: heptane/ethyl acetate=24/1→5/1), 849 mg of thetitle compound (14) was obtained.

Step 4: Synthesis of Compound (15)

Under an argon atmosphere, after a tetrahydrofuran solution (30 mL) ofthe compound (8) (928 mg, 3.00 mmol) was cooled with ice, sodium hydride(60% oil, 144 mg, 3.60 mmol) was added thereto. After the reactionliquid was heated to room temperature and stirred for 30 minutes, thecompound (14) (784 mg, 4.50 mmol) was added thereto. After the reactionliquid was heated to 40° C. and the raw material disappeared, water wasadded in the reaction liquid, and the organic layer was extracted usingethyl acetate. After the organic layer was washed with water andsaturated saline water and dried with anhydrous sodium sulphate, thesolvent was distillated under reduced pressure. The residue was refinedby column chromatography (developing solvent:chloroform→chloroform/methanol=50/1). Methanol was added to the refinedproduct, the refined product was suspended and washed, and theprecipitate was filtered. By drying the cake under reduced pressure, 583mg of the title compound (15) was obtained.

Step 5: Synthesis of PBQ3.1

Under an argon atmosphere, after a dichloromethane solution (5.0 mL) ofthe compound (15) (329 mg, 1.00 mmol) was cooled down to −40° C., borontribromide (1.0 M dichloromethane solution, 5.00 mL, 5.00 mmol) wasadded dropwise. The reaction liquid was heated to 5° C., and stirred allnight. After the reaction liquid was neutralized by adding a 1N sodiumhydroxide aqueous solution and sodium hydrogen carbonate under ice coldconditions, the organic layer was extracted with ethyl acetate. Afterthe organic layer was washed with water and saturated saline water anddried with anhydrous sodium sulphate, the solvent was distillated underreduced pressure. The residue was refined by column chromatography(developing solvent: chloroform/methanol=99/1→17/1). Methanol was addedto the refined product, the refined product was suspended and washed,and the precipitate was filtered. By drying the cake under reducedpressure, 147 mg of PBQ3.1 was obtained.

PBQ3.1: ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 10.09 (s, 1H), 8.26 (d, J=1.83Hz, 1H), 8.12 (d, J=8.70 Hz, 1H), 7.82 (d, J=9.16 Hz, 1H), 7.66 (d,J=8.70 Hz, 1H), 7.61 (dd, J=9.16 Hz, 2.29 Hz, 1H), 7.30 (dd, J=9.16 Hz,2.75 Hz, 1H), 7.13 (d, J=16.03 Hz, 1H), 7.12 (d, J=2.75 Hz, 1H), 7.05(d, J=16.03 Hz, 1H), 6.67 (d, J=8.70 Hz, 1H), 3.07 (s, 6H).

Synthesis Embodiment 29 Synthesis of(E)-2-(4-(6-(methylamino)pyridine-3-yl)buta-1-en-3-ynyl)quinoline-6-ol(PBQ3.2)

PBQ3.2 was synthesized according to the following synthesis scheme:

Step 1: Synthesis of Compound (8)

Under an argon atmosphere, after a tetrahydrofuran solution (600 mL) of6-methoxy-2-methylquinoline (7) (43.0 g, 248 mmol) was cooled down to−70′C, tert-butyllithium (1.61M hexane solution, 200 mL, 322 mmol) wasadded dropwise. The reaction liquid was stirred for 1 hour, and diethylchlorophosphate (59.9 g, 347 mmol) was added dropwise. The reactionliquid was stirred for 1 hour, and, after water was added and thereaction liquid was stirred all night, the reaction liquid was extractedwith ethyl acetate. After the organic layer was washed with saturatedsaline water and dried with anhydrous sodium sulphate, the solvent wasdistillated under reduced pressure. By refining the residue by columnchromatography (developing solvent: ethyl acetate→ethylacetate/methanol=19/1), 27.2 g of the title compound (8) was obtained.

Step 2: Synthesis of Compound (10)

Under an argon atmosphere, after a tetrahydrofuran solution (30 mL) ofthe compound (8) (928 mg, 3.00 mmol) was cooled with ice, sodium hydride(60% oil, 144 mg, 3.60 mmol) was added thereto. After the reactionliquid was heated to room temperature and stirring for 30 minutes, thecompound (9) (937 mg, 3.60 mmol) was added. After the reaction liquidwas heated to 40° C. and the raw material disappeared, water was addedin the reaction liquid, and the reaction liquid was extracted with ethylacetate. After the organic layer was washed with water and saturatedsaline water and dried with anhydrous sodium sulphate, the solvent wasdistillated under reduced pressure. By refining the residue by columnchromatography (developing solvent: heptane/ethyl acetate=7/1→3/1), 580mg of the title compound (10) was obtained.

Step 3: Synthesis of PBQ3.2

Under an argon atmosphere, after a dichloromethane solution (7.0 mL) ofthe compound (10) (575 mg, 1.38 mmol) was cooled down to −40° C., borontribromide (1.0 M dichloromethane solution, 11.1 mL, 11.1 mmol) wasadded dropwise. The reaction liquid was heated to 5° C., and stirred allnight. After the reaction liquid was neutralized by adding a 1N sodiumhydroxide aqueous solution and a sodium hydrogen carbonate solutionunder ice cold conditions, the precipitate was filtered. The cake wasrefined by column chromatography (developing solvent:chloroform/methanol=99/1→19/1). Methanol was added to the refinedproduct, the refined product was suspended and washed, and theprecipitate was filtered. By drying the cake under reduced pressure, 110mg of PBQ3.2 was obtained.

PBQ3.2: ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 10.09 (s, 1H), 8.18 (d, J=2.29Hz, 1H), 8.11 (d, J=8.70 Hz, 1H), 7.82 (d, J=9.16 Hz, 1H), 7.66 (d,J=8.70 Hz, 1H), 7.48 (dd, J=8.70 Hz, 2.29 Hz, 1H), 7.30 (dd, J=9.16 Hz,2.75 Hz, 1H), 7.12 (d, J=2.75 Hz, 1H), 7.11 (d, J=16.03 Hz, 1H),7.02-7.07 (m, 1H), 7.04 (d, J=16.03 Hz, 1H), 6.47 (d, J=8.70 Hz, 1H),2.80 (d, J=4.58 Hz, 3H).

Synthesis Embodiment 30 Synthesis of2-((1E,3E)-4-(4-aminophenyl)buta-1,3-dienyl)benz[d]thiazole-6-ol (pre2)

pre2 was synthesized according to the following synthesis scheme:

Step 1: Synthesis of Compound (6)

Under an argon atmosphere, after a tetrahydrofuran solution (75 mL) ofdiisopropylamine (5.06 g, 50.0 mmol) was cooled down to −50° C.,n-butyllithium (1.6 M hexane solution, 31.2 mL, 50.0 mmol) was addeddropwise. The reaction liquid was cooled down to −65° C., and atetrahydrofuran solution (25 mL) of 6-methoxy-2-methylbenzothiazole(5)(4.48 g, 25.0 mmol) was added dropwise. Diethyl chlorophosphate (4.31g, 25.0 mmol) was added dropwise to the reaction liquid. After thedisappearance of the raw material, the reaction liquid was added in 100mL of a 1M hydrogen chloride solution, and the organic layer wasextracted with chloroform. The organic layer was dried with anhydroussodium sulphate, and the solvent was distillated under reduced pressure.By refining the residue by column chromatography (developing solvent:chloroform), 6.30 g of the title compound (6) was obtained.

Step 2: Synthesis of Compound (7)

Under an argon atmosphere, after a tetrahydrofuran solution (10 mL) ofthe compound (6) (380 mg, 1.21 mmol) was cooled with ice, sodium hydride(60% oil, 48 mg, 1.20 mmol) was added thereto. After the reaction liquidwas heated to room temperature and stirred for 30 minutes,4-nitrocinnamaldehyde (180 mg, 1.02 mmol) was added. After thedisappearance of the raw material, the reaction liquid was added inwater and stirred, and the precipitate was filtered. Toluene was addedto the cake, and the solvent was distillated under reduced pressure, andsuspended and washed with chloroform. By filtering and drying underreduced pressure the precipitate, 275 mg of the title compound (7) wasobtained.

Step 3: Synthesis of Compound (8)

Acetic acid (5.1 mL), iron (212 mg, 3.80 mmol) and 12N hydrochloric acid(1.1 mL) were added in an ethanol solution (5.1 mL) of the compound (7)(271 mg, 0.80 mmol), and the resultant solution was stirred all night.The reaction liquid was added dropwise in a sodium hydroxide aqueoussolution under ice cold conditions, and, after chloroform was added, thereaction liquid was filtered. After the filtrate was extracted withchloroform and the organic layer was dried with anhydrous sodiumsulphate, the solvent was distillated under reduced pressure. Byrefining the residue by column chromatography (developing solvent:chloroform), 165 mg of the title compound (8) was obtained.

Step 4: Synthesis of2-((1E,3E)-4-(4-aminophenyl)buta-1,3-dienyabenz[d]thiazole-6-ol (pre2)

Under an argon atmosphere, after a dichloromethane solution (2.6 mL) ofthe compound (8) (160 mg, 0.52 mmol) was cooled down to −78° C., borontribromide (1.0M dichloromethane solution, 2.60 mL, 2.60 mmol) was addeddropwise. The reaction liquid was heated to room temperature, andstirred all night. After the reaction liquid was made alkaline by addinga 1N sodium hydroxide aqueous solution under ice cold conditions, theresultant liquid was filtered. The filtrate was neutralized by adding 1Nhydrochloric acid and sodium hydrogen carbonate, and the precipitate wasfiltered. Chloroform was added to the cake, the resultant mixture wassuspended and washed, and the precipitate was filtered. Methanol wasadded to the cake, the resultant mixture was suspended and washed, andthe precipitate was filtered. By drying the cake under reduced pressure,120 mg of the title compound was obtained.

pre2: ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 9.80 (s, 1H), 7.69 (d, J=8.70 Hz,1H), 7.31 (d, J=2.29 Hz, 1H), 7.25 (d, J=8.70 Hz, 2H), 7.20 (dd, J=16.03Hz, 9.16 Hz, 1H), 6.92 (dd, J=8.70 Hz, 2.29 Hz, 1H), 6.81-6.91 (m, 2H),6.81 (d, J=16.03 Hz, 1H), 6.56 (d, J=8.70 Hz, 2H), 5.52 (s, 2H)

Synthesis Embodiment 31 Synthesis of5-((1E,3E)-4-(6-(tert-butyldimethylsilyloxy)benz[d]thiazole-2-yl)buta-1,3-dienyl)pyridine-2-amine(pre3)

pre3 was synthesized according to the following synthesis scheme:

Step 1: Synthesis of Compound (14)

Under an argon atmosphere, after a dichloromethane solution (2.9 mL) ofthe compound (12) (184 mg, 0.57 mmol) was cooled down to −78° C., borontribromide (1.0M dichloromethane solution, 2.85 mL, 2.85 mmol) was addeddropwise. The reaction liquid was heated to room temperature, andstirred all night. The reaction liquid was neutralized by adding a 1Nsodium hydroxide aqueous solution and sodium hydrogen carbonate underice cold conditions, and the solvent was distillated under reducedpressure. The residue was suspended and washed with water. Theprecipitate was filtered and dried under reduced pressure, therebygiving 154 mg of the title compound (14).

Step 2: Synthesis of5-((1E,3E)-4-(6-(tert-butyldimethylsilyloxy)benz[d]thiazole-2-yl)buta-1,3-dienyl)pyridine-2-amine(pre3)

Under an argon atmosphere, imidazole (72.6 mg, 1.066 mmol) andt-butyldimethylchlorosilane (73.5 mg, 0.489 mmol) were added in adimethylsulfoxide solution (2.58 mL) of the compound (14) (90.0 mg,0.305 mmol), and the resultant solution was stirred all night. Water wasadded in the reaction liquid, and the reaction liquid was extracted withethyl acetate. After the organic layer was washed with saturated salinewater and dried with anhydrous sodium sulphate, the solvent wasdistillated under reduced pressure. By refining the residue by columnchromatography (developing solvent:chloroform→chloroform/methanol=100/7), 52 mg of the title compound wasobtained.

pre3: ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 8.04 (d, J=2.29 Hz, 1H), 7.77 (d,J=8.07 Hz, 1H), 7.68 (dd, J=8.70 Hz, 2.29 Hz, 1H), 7.53 (d, J=2.29 Hz,1H), 7.28 (dd, J=15.57 Hz, 10.08 Hz, 1H), 6.99 (dd, J=8.70 Hz, 2.75 Hz,1H), 6.88-6.96 (m, 1H), 6.86 (d, J=15.57 Hz, 1H), 6.85 (d, J=15.57 Hz,1H), 6.47 (d, J=8.70 Hz, 1H), 6.35 (s, 2H), 0.98 (s, 9H), 0.23 (s, 6H)

Synthesis Embodiment 32 Synthesis of2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dienyl)-3-ethyl-6-hydroxy-benz[d]thiazole-3-ium(pre6)

The synthesis was performed in a method similar to the synthesis methodsof synthesis example 5 and PBB5 above.

Synthesis Embodiment 33 Synthesis of(E)-5-(4-(6-(tert-butyldimethylsilyloxy)benz[d]thiazole-2-yl)buta-3-en-1-ynyl)pyridine-2-amine (pre11)

pre11 was synthesized according to the following synthesis scheme:

Step 1: Synthesis of6-(t-butyldimethylsilyloxy)benzothiazole-2-carboxylic Acid Ethyl (8)

A DMF solution (3 mL) of t-butyldimethylchlorosilane (0.94 g, 6.2 mmol)was added in a DMF solution (10 mL) of6-hydroxy-benzothiazole-2-carboxylic acid ethyl (1.27 g, 5.69 mmol) andimidazole (0.5 g, 7.34 mmol), and, after the resultant solution wasstirred at room temperature for 16 hours, water was added, and theresultant liquid was extracted with ethyl acetate. After the extractedliquid was washed with water, the resultant liquid was dried withanhydrous sodium sulphate, and the solvent was distillated at reducedpressure. The resulting residue was refined by silica gel columnchromatography, and6-(t-butyldimethylsilyloxy)benzothiazole-2-carboxylic acid ethyl wasobtained as a brown liquid (0.97 g, 2.9 mmol).

¹H-NMR (400 MHz, CDCl₃) δ ppm: 8.09 (d, J=8.8 Hz, 1H), 7.35 (d, J=2.4Hz, 1H), 7.09 (dd, J=8.8 Hz, 2.4 Hz), 4.54 (q, J=7.2 Hz, 2H), 1.48 (t,J=7.2 Hz, 3H), 1.01 (s, 9H) (s, 6H)

Step 2: Synthesis of[6-(t-butyldimethylsilyloxy)benzothiazole-2-yl]methanol (9)

A THF solution (20 mL) of lithium aluminium hydride (87 mg, 2.3 mmol)was cooled down to −15° C., and a THF solution (10 mL) of6-(t-butyldimethylsilyloxy)benzothiazole-2-carboxylic acid ethyl (0.77g, 2.3 mmol) was added dropwise. After the resultant solution wasstirred at the same temperature for 1 hour, lithium aluminium hydride(72.5 mg, 1.91 mmol) was added thereto, and the resultant solution wasstirred for 30 more minutes. Water (0.16 mL) was added in the resultantsolution, and, after stirring for a while, a 5M sodium hydroxide aqueoussolution (0.16 mL) was added in the solution, followed by water (0.48mL), and, after stirring, the insoluble matter was filtered usingcelite. The filtrate was condensed under reduced pressure, the residuewas refined by silica gel column chromatography, and[6-(t-butyldimethylsilyloxy)benzothiazole-2-yl] methanol was obtained asa brown liquid (0.22 g, 0.74 mmol).

¹H-NMR (400 MHz, CDCl₃) δ ppm: 7.84 (d, J=8.8 Hz, 1H), 7.33 (d, J=2.4Hz, 1H), 7.01 (dd, J=8.8 Hz, 2.4 Hz), 5.05 (br s, 2H) 2.78 (br s, 1H),1.03 (s, 9H), 0.25 (s, 6H)

Step 3: Synthesis of6-(t-butyldimethylsilyloxy)benzothiazole-2-carboxaldehyde (6)

A manganese dioxide powder (1.2 g) was added in a dichloromethanesolution (30 mL) of[6-(t-butyldimethylsilyloxy)benzothiazole-2-yl]methanol (0.22 g, 0.74mmol), and the resultant solution was stirred for 2.5 hours at 40° C.and for 16 hours at room temperature. The insoluble matter was filteredusing celite, and the filtrate was condensed under reduced pressure. Theresulting residue was refined by silica gel column chromatography, and6-(t-butyldimethylsilyloxy)benzothiazole-2-carboxaldehyde was obtainedas a brown liquid (71.0 mg, 0.242 mmol).

¹H-NMR (400 MHz, CDCl₃) δ ppm: 10.11 (s, 1H), 8.09 (d, J=8.8 Hz, 1H),7.37 (d, J=2.4 Hz, 1H), 7.13 (dd, J=8.8 Hz, 2.4 Hz), 1.01 (s, 9H), 0.27(s, 6H)

Step 4: Synthesis of2-[(E)-2-bromoethenyl]-6-(t-butyldimethylsilyloxy)benzothiazole (7)

(Bromodifluormethyl) triphenylphosphonium bromide (48.2 mg, 0.11 mmol)was suspended in THF (including THF as a stabilizer, 3 mL), theresultant mixture was cooled down to −78° C., n-butyllithium (1.6Mhexane solution, 0.15 mL) was added thereto, and the resultant mixturewas stirred for 1 hour. Next, a THF solution (2 mL) of6-(t-butyldimethylsilyloxy)benzothiazole-2-carboxaldehyde (20.2 mg,0.0688 mmol) was added, and the resultant mixture was stirred at forapproximately 30 minutes at −78° C., and for approximately 1.5 hours atO° C. A saturated ammonium chloride aqueous solution was added in thereaction liquid (3 mL), and the resultant liquid was stirred for 10minutes, and, after water and ethyl acetate were added, the resultantliquid was separated. After the organic layer was washed with saturatedsaline water, dried with anhydrous sodium sulphate, and condensed underreduced pressure, the resultant product was refined by silica gel columnchromatography, and a mixture of 2-RE)-2-bromoethenyl1-6-(t-butyldimethylsilyloxy)benzothiazole and BHT was obtained as ayellow liquid (7.0 mg). When the content of BHT and the title compoundis to be calculated from the intensity ratio of 11-1-NMR signal, it isestimated that approximately 5.5 mg (0.015 mmol) of the title compoundis contained.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 7.84 (d, J=8.8 Hz, 1H), 7.35 (d, J=14.0Hz, 1H), 7.29 (d, J=14.0 Hz, 1H), 7.25 (d, J=2.4 Hz, 1H), 7.00 (dd,J=8.8 Hz, 2.4 Hz), 1.00 (s, 9H), 0.26 (s, 6H)

Step 5: Synthesis of(E)-5-(4-(6-(t-butyldimethylsilyloxy)benzothiazole-2-yl)-3-butene-1-ynyl)pyridine-2-amine(1)

A mixture of2-[(E)-2-bromoethenyl]-6-(t-butyldimethylsilyloxy)benzothiazole and BHT(18.1 mg, including 13.5 mg of2-[(E)-2-bromoethenyl]-6-(t-butyldimethylsilyloxy)benzothiazole),2-amino-5-ethynylpyridine (8.7 mg, 0.074 mmol), cuprous iodide (0.7 mg),and dichlorobis (triphenylphosphine) palladium (2 mg) were added in aliquid mixture of THF (1 mL) and triethylamine (1 mL), and the resultantmixture was stirred at 70° C. for 4 hours. After ethyl acetate wasadded, the insoluble matter was filtered, and, after the filtrate wascondensed under reduced pressure and refined by silica gel columnchromatography, a mixture of the title compound and its (Z)-isomer wasobtained as a yellow-brown amorphous solid (9.4 mg). E/Z=approximately85/15 (¹H-NMR).

pre11: ¹H-NMR (400 MHz, CDCl₃) δ ppm: 8.24 (br d, J=2.0 Hz, 1H), 7.84(d, J=8.8 Hz, 1H), 7.53 (dd, J=8.4 Hz, 2.0 Hz), 7.26 (d, J=2.4 Hz, 1H),7.16 (d, J=16.0 Hz, 1H), 6.98 (dd, J=8.8 Hz, 2.4 Hz), 6.73 (d, J=16.0Hz, 1H), 6.47 (dd, J=8.4 Hz, 0.4 Hz), 4.70 (s, 2H), 1.01 (s, 9H), 0.23(s, 6H)

Synthesis Embodiment 34 Synthesis of(E)-tert-butyl(2-(4-(6-aminopyridine-3-yl)buta-1-en-3-ynyl)benz[d]thiazole-6-yl)methylcarbamate(pre12)

pre12 was synthesized according to the following synthesis scheme:

2-amino-5-ethynylpyridine (compound A) was synthesized from2-amino-5-iodopyridine, as shown in the above scheme.

From 2-amino-5-ethynylpyridine (compound A) (0.14 g, 1.2 mmol) and2-((E)-2-bromoetheny0-6-((tert-butoxycarbonylamino)methyl)benzothiazole(0.22 g, 0.60 mmol), the title compound was obtained in the sameprocedures as in step 5 of synthesis example 33 above (181.7 mg, 0.447mmol).

Pre12: ¹H-NMR (400 MHz, CDCl₃) δ ppm: 8.25 (d, J=1.6 Hz, 1H), 7.94 (d,J=8.4 Hz, 1H), 7.77 (br s, 1H), 7.53 (dd, J=8.8 Hz, 2.4 Hz, 1H), 7.39(br d, J=8.4 Hz, 1H), 7.17 (d, J=16.0 Hz, 1H), 6.83 (d, J=16.0 Hz, 1H),6.47 (dd, J=8.8 Hz, 0.8 Hz, 1H), 4.9 (br, 1H), 4.66 (s, 2H), 4.44 (br d,J=6.4 Hz, 2H), 1.47 (s, 9H)

Synthesis Embodiment 35-1 Synthesis of 2-(2-((1E,3E)-4-(6-(methylamino)pyridine-3-yl)buta-1,3-dienyl)benz[d]thiazole-6-yloxy)-2-hydroxymethyl-ethyl4-methylbenzenesulfonate (Analog of pre21)

An analog of pre21 was synthesized according to the following synthesisscheme:

Step 1: Synthesis of Compound (23)

Under an argon atmosphere, after pyridine (7910 mg, 100.0 mmol) wasadded in a dichloromethane solution (10 mL) of2,2-dimethyl-1,3-dioxolane-4-methanol (22) (1322 mg, 10.0 mmol) and theresultant solution was cooled with ice, p-oluenesulfonylchloride (2860mg, 15.0 mmol) and N,N-dimethylaminopyridine (12 mg, 0.10 mmol) wereadded, and the resultant solution was stirred. After the disappearanceof the raw material, water was added in the reaction liquid, and thereaction liquid was extracted using ethyl acetate. After the organiclayer was washed with a hydrochloric acid aqueous solution, a sodiumhydrogen carbonate aqueous solution and saturated saline water and driedwith anhydrous sodium sulphate, the solvent was distillated underreduced pressure, and 2560 mg of the title compound (23) was obtained.

Step 2: Synthesis of Compound (24)

4N hydrochloric acid/dioxane (2.5 mL) was added in a methanol solution(7.5 mL) of the compound (23) (1432 mg, 5.00 mmol), and the resultantsolution was stirred. After the disappearance of the raw material, thereaction liquid was distillated under reduced pressure, and, by refiningthe residue by column chromatography (developing solvent: heptane/ethylacetate=1/4→ethyl acetate), 1027 mg of the title compound (24) wasobtained.

Step 3: Synthesis of Compound (25)

Under an argon atmosphere, imidazole (272 mg, 4.00 mmol) was added in atetrahydrofuran solution (4.0 mL) of the compound (24) (985 mg, 4.00mmol), and the resultant solution was cooled with ice. A tetrahydrofuransolution (4.0 mL) of t-butyldimethylchlorosilane (603 mg, 4.00 mmol) wasadded dropwise to the reaction liquid. After the disappearance of theraw material, water was added in the reaction liquid, and the organiclayer was extracted with ethyl acetate. After the organic layer waswashed with water and saturated saline water and dried with anhydroussodium sulphate, the solvent was distillated under reduced pressure. Byrefining the residue by column chromatography (developing solvent:heptane/ethyl acetate=7/1→4/1), 1182 mg of the title compound (25) wasobtained.

Step 4: Synthesis of Compound (27)

Under an argon atmosphere, after a N,N-dimethylformamide solution (11mL) of the compound (26) (which had been synthesized in the previoustest preparation report) (696 mg, 2.25 mmol) was cooled with ice, sodiumhydride (60% oil, 360 mg, 9.00 mmol) was added in the resultantsolution. The reaction liquid was heated to room temperature and stirredfor 30 minutes. After the reaction liquid was cooled with ice and methyliodide (1277 mg, 9.00 mmol) was added thereto, the reaction liquid washeated to room temperature. After the disappearance of the raw material,the reaction liquid was added in water and stirred, and the precipitatewas filtered. By refining the cake by column chromatography (developingsolvent: chloroform→chloroform/methanol=99/1), 554 mg of the titlecompound (27) was obtained.

Step 5: Synthesis of Compound (28)

Under an argon atmosphere, after a dichloromethane solution (13 mL) ofthe compound (27) (550 mg, 1.63 mmol) was cooled down to −70° C., borontribromide (1.0M dichloromethane solution, 16.3 mL, 16.30 mmol) wasadded dropwise. The reaction liquid was heated to 9° C., and stirred allnight. After the reaction liquid was cooled with ice and neutralized byadding sodium hydroxide aqueous solution, the organic layer wasdistillated under reduced pressure. The precipitate was filtered, washedwith water, and dried under reduced pressure, and 484 mg of the titlecompound (28) was obtained.

Step 6: Synthesis of Compound (29)

Under an argon atmosphere, the compound (25) (721 mg, 2.00 mmol) andtriphenylphosphine (525 mg, 2.00 mmol) were added in a tetrahydrofuransolution (10.0 mL) of the compound (28) (323 mg, 1.00 mmol), and theresultant solution was cooled with ice. Diisopropyl azodicarboxylate(404 mg, 2.00 mmol) was added dropwise to the reaction liquid. After thereaction liquid was heated to room temperature and stirred all night,the reaction liquid was distillated under reduced pressure. By refiningthe residue by column chromatography (developing solvent: heptane/ethylacetate=3/1→1/2), 270 mg of the title compound (29) was obtained.

Step 7: Synthesis of Compound (5)

4N hydrochloric acid/dioxane (1.5 mL) was added in a tetrahydrofuransolution (4.5 mL) of the compound (29) (200 mg, 0.30 mmol), and theresultant solution was stirred. After the disappearance of the rawmaterial, the reaction liquid was cooled with ice and neutralized with asodium hydrogen carbonate aqueous solution, and then the reaction liquidwas extracted with ethyl acetate. After the organic layer was washedwith water and saturated saline water and dried with anhydrous sodiumsulphate, the solvent was distillated under reduced pressure. Byrefining the residue by column chromatography (developing solvent:heptane/ethyl acetate=1/1→1/4), 134 mg of the title compound (5) wasobtained.

Compound (5): ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 8.21 (d, J=2.29 Hz, 1H),7.79 (dd, J=9.16 Hz, 2.29 Hz, 1H), 7.75 (d, J=9.16 Hz, 1H), 7.73 (d,J=8.24 Hz, 2H), 7.52 (d, J=2.75 Hz, 1H), 7.39 (d, J=8.24 Hz, 2H), 7.31(dd, J=14.78 Hz, 10.08 Hz, 1H), 6.85-7.06 (m, 4H), 6.70 (d, J=9.16 Hz,1H), 5.07 (t, J=5.50 Hz, 1H), 4.53-4.60 (m, 1H), 4.20-4.35 (m, 2H),3.52-3.63 (m, 2H), 3.07 (s, 6H), 2.35 (s, 3H).

Synthesis Embodiment 35-2 Synthesis of3-(2-((1E,3E)-4-(6-(methylamino)pyridine-3-yl)buta-1,3-dienyl)benz[d]thiazole-6-yloxy)-2-(tetrahydro-2H-pyran-2-yloxy)propyl4-methylbenzenesulfonate (pre21)

pre21 can be synthesized by the same method as that of synthesis example35-1 above.

Synthesis Embodiment 36 Synthesis of(E)-3-(2-(4-(6-(methylamino)pyridine-3-yl)buta-1-en-3-ynyl)benz[d]thiazole-6-yloxy)-2-(tetrahydro-2H-pyran-2-yloxy)propyl4-methylbenzenesulfonate (pre22)

pre22 can be synthesized by a similar method to those of synthesisexamples 22 to 25 above.

Synthesis Embodiment 37 Synthesis of tert-butyl5-((1E,3E)-4-(6-(ethoxymethoxy)benz[d]thiazole-2-yl)buta-1,3-dienyl)-6-nitropyridine-2-yl(methyl)carbamate (pre23)

pre23 can be synthesized by a similar method to those of synthesisexamples 22 to 25 above.

Synthesis Embodiment 38 Synthesis of (E)-tert-butyl5-(4-(6-(ethoxymethoxy)benz[d]thiazole-2-yl)buta-3-en-1-ynyl)-6-nitropyridine-2-yl(methyl)carbamate(pre24)

pre24 can be synthesized by a similar method to those of synthesisexamples 22 to 25 above.

Synthesis Embodiment 39 Synthesis of tert-butyl5-((1E,3E)-4-(5-(ethoxymethoxy)benzofuran-2-yl)buta-1,3-dienyl)-6-nitropyridine-2-yl(methyl)carbamate(pre25)

pre25 can be synthesized by a similar method to those of synthesisexamples 22 to 25 above.

Synthesis Embodiment 40 Synthesis of (E)-tert-butyl5-(4-(5-(ethoxymethoxy)benzofuran-2-yl)buta-3-en-1-ynyl)-6-nitropyridine-2-yl(methyl)carbamate(pre26)

pre26 can be synthesized by a similar method to those of synthesisexamples 22 to 25 above.

Synthesis of Radioisotope-Labeled Compound Synthesis Embodiment 41Synthesis of4-((1E,3E)-4-(benz[d]thiazole-2-yl)buta-1,3-dienyl)-N-[¹¹C]methyl-N-methylaniline([¹¹C]PBB1)

[¹¹C]PBB1 was synthesized according to the same method as the methodsshown in following synthesis examples 42 and 43.

Synthesis Embodiment 42 Synthesis of2-((1E,3E)-4-(4-([¹¹C]methylamino)phenyl)buta-1,3-dienyl)benz[d]thiazole-6-ol([¹¹C]PBB2)

[11C]methyltriflate was added in an acetone solution (500 mL), contained2-((1E,3E)-4-(4-aminophenyl)buta-1,3-dienyl)benz[d]thiazole-6-ol (pre2)(0.5 to 0.8 mg), at room temperature. Under a nitrogen atmosphere,acetone was removed at 80° C., and a 70% acetonitrile aqueous solution(800 μL) was added. The liquid mixture was moved to a HPLC purifyingcontainer (HPLC: CAPCELL PAK C18 column, 10 mm×250 mm, SHISEIDO; mobilephase, acetonitrile/water/triethylamine=700/300/1, 6 mL/minute). Thefractions to match [¹¹C]PBB2 were collected in a flask, which contained,in ethanol (300 μL), 25% ascorbic acid (100 μL) and Tween80 (75 μL), andthe solvent was distillated under reduced pressure. The residue wasdissolved in a physiological saline water (3 mL, pH 7.4), and [¹¹C]PBB2(640-1340 GBq) was obtained as an injection solution.

Synthesis Embodiment 43 Synthesis of2-((1E,3E)-4-(6-([¹¹C]methylamino)pyridine-3-yl)buta-1,3-dienyl)benz[d]thiazole-6-ol([¹¹C]PBB3)

Iodo [¹¹C]methane was added, at room temperature, in a DMSO solution(300 μL), which contained5-((1E,3E)-4-(6-(tert-butyldimethylsilyloxy)benz[d]thiazole-2-yl)buta-1,3-dienyl)pyridine-2-amine(pre3) (1.5 to 2 mg) and potassium hydroxide (10 mg). The reactionliquid mixture was heated to 125° C. and maintained for 5 minutes. Afterthe reaction container was cooled down, an aqueous solution (600 μL) ofa tetra-n-butylammoniumfluoride hydrate (5 mg) μ was added, and theprotecting group was removed. After that, a HPLC solvent (500 μL) wasadded. The liquid mixture was moved to an HPLC purifying container(HPLC: CAPCELL PAK C18 column, 10 mm×250 mm, acetonitrile/50 mM ammoniumformate=4/6, 6 mL/minute). The fractions to match [¹¹C]PBB3 werecollected in a flask, which contained, in ethanol (300 μL), 25% ascorbicacid (100 μL) and Tween80 (75 μL), and the solvent was distillated underreduced pressure. The residue was dissolved in physiological salinewater (3 mL, pH 7.4), and [¹¹C]PBB3 (970-1990 GBq) was obtained as aninjection solution.

Synthesis Embodiment 44 Synthesis of 2-((1E,3E)-4-(6-(^([11)C]methylamino)pyridine-3-yl)buta-1,3-dienyl)benz[d]thiazole-5,6-diol([¹¹C]PBB4)

[¹¹C]PBB4 was synthesized according to the same method as the methodsshown in synthesis examples 42 and 43 above.

Synthesis Embodiment 45 Synthesis of2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dienyl)-3-ethyl-6-[¹¹C]methoxybenzo[d]thiazole-3-ium ([11C] mPBB5)

Iodo[¹¹C]methane was added, at −15° C., in a DMF (300 μL) solution,which contained2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dienyl)-3-ethyl-6-hydroxy-benz[d]thiazole-3-ium(pre6) (0.8 to 0.9 mg) and sodium hydride (0.3 mg). The reaction liquidmixture was heated to 80° C., and maintained for 5 minutes. A 60%methanol aqueous solution (800 μL) was added, and the resultant mixturewas moved to a HPLC purifying container (HPLC: CAPCELL PAK C18 column,10 mm×250 mm, mobile phase, methanol/water/trifluoroaceticacid=600/400/0.1, 4 mL/minute). The fractions to match [¹¹C]mPBB5 werecollected in a flask, which contained, in ethanol (300 μL), 25% ascorbicacid (100 μL) and Tween80 (75 μL), and the solvent was distillated underreduced pressure. The residue was dissolved in physiological salinewater (3 mL, pH 7.4), and [¹¹C]mPBB5 (300-560 GBq) was obtained as aninjection solution.

Synthesis Embodiment 46 Synthesis of(E)-2-(4-(4-(N-[¹¹C]methyl-N-methylamino)phenyl)buta-1-en-3-ynyl)benz[d]thiazole-6-ol([¹¹C]PBB2.1)

[¹¹C]PBB2.1 was synthesized from pre7 by the same method as the methodsshown in synthesis examples 42 and 43 above.

Synthesis Embodiment 47 Synthesis of(E)-2-(4-(4-([¹¹C]methylamino)phenyl(buta-1-en-3-ynyl)benz[d]thiazole-6-ol([¹¹C]PBB2.2)

[¹¹C]PBB2.2 was synthesized from pre8 by the same method as the methodsshown in synthesis examples 42 and 43 above.

Synthesis Embodiment 48 Synthesis of(E)-2-(4-(6-(N-[¹¹C]methyl-N-methylamino)pyridine-3-yl)buta-1-en-3-ynyl)benz[d]thiazole-6-ol([¹¹C]PBB3.1)

[¹¹C]PBB3.1 was synthesized by the same method as the methods shown insynthesis examples 42 and 43 above.

Synthesis Embodiment 49 Synthesis of(E)-2-(4-(6-([¹¹C]methylamino)pyridine-3-yl)buta-1-en-3-ynyl)benz[d]thiazole-6-ol([¹¹C]PBB3.2)

[¹¹C]PBB3.2 was synthesized from pre11 by the same method as the methodsshown in synthesis examples 42 and 43 above.

Synthesis Embodiment 50 Synthesis of(E)-5-(4-(6-(aminomethyl)benz[d]thiazole-2-yl)buta-3-en-1-ynyl)-N-[¹C]methylpyridine-2-amine ([¹¹C]PBB3.2N)

[¹¹C]PBB3.2N was synthesized from pre12 by the same method as themethods shown in synthesis examples 42 and 43 above.

Synthesis Embodiment 51 Synthesis of(2-((1E,3E)-4-(4-aminophenyl)buta-1,3-dienyl)-6-[¹¹C]methoxybenzo[d]thiazole-5-ol([¹¹C]Core1-4)

[¹¹C]Core1-4 was synthesized by the same method as the method shown insynthesis example 45 above.

Synthesis Embodiment 52 Synthesis ofN-(4-((1E,3E)-4-(5-methoxy-6-[¹¹C]methoxybenzo[d]thiazole-2-yl)buta-1,3-dienyl)phenyl)acetamide([¹¹C]Core1-5)

[¹¹C]Core1-5 was synthesized by the same method as the method shown insynthesis example 45 above.

Synthesis Embodiment 53 Synthesis of(3-(4-(1E,3E)-4-(5-methoxy-6-[¹¹C]methoxybenzo[d]thiazole-2-yl)buta-1,3-dienyl)phenylamino)propan-1-ol([C]Core1-11)

[¹¹C]Core1-11 was synthesized by the same method as the method shown insynthesis example 45 above.

Synthesis Embodiment 54 Synthesis of4-((1E,3E)-4-(5-methoxy-6-[¹¹C]methoxybenzo[d]thiazole-2-yl)buta-1,3-dienyl)-N-isopropylaniline([¹¹C]Core1-15)

[¹¹C]Core1-15 was synthesized by the same method as the method shown insynthesis example 45 above.

Synthesis Embodiment 55 Synthesis of4-((1E,3E)-4-(5-methoxy-6-[¹¹C]methoxybenzo[d]thiazole-2-yl(buta-1,3-dienyl)-N-(hepta-1,6-diene-4-ypaniline([¹¹C]Core1-20)

[¹¹C]Core1-20 was synthesized by the same method as the method shown insynthesis example 45 above.

Synthesis Embodiment 56 Synthesis ofN-(5-((E,3E)-4-(5-methoxy-6-[¹¹C]methoxybenzo[d]thiazole-2-yl)buta-1,3-dienyl)pyridine-2-yl)acetamide([¹¹C]Core2-9)

[¹¹C]Core2-9 was synthesized by the same method as the method shown insynthesis example 45 above.

Synthesis Embodiment 57 Synthesis of3-(5-((1E,3E)-4-(5-methoxy-6-[¹¹C]methoxybenzo[d]thiazole-2-yl)buta-1,3-dienyl)pyridine-2-ylamino)propan-1-ol([¹¹C] Core2-10)

[¹¹C]Core2-10 was synthesized by the same method as the method shown insynthesis example 45 above.

Synthesis Embodiment 58 Synthesis ofN,N-diallyl-5-((1E,3E)-4-(5-methoxy-6-[¹¹C]methoxybenzo[d]thiazole-2-yl)buta-1,3-dienyl)pyridine-2-amine([¹¹C]Core2-14)

[¹¹C]Core2-14 was synthesized by the same method as the method shown insynthesis example 45 above.

Synthesis Embodiment 59-1 Synthesis of1-[¹⁸F]fluoro-2-(24(1E,3E)-4-(6-(dimethylamino)pyridine-3-yl)buta-1,3-dienyl)benz[d]thiazole-6-yloxy)-hydroxymethyl-ethane(Analog of [¹⁸F]F0-PBB3)

A [¹⁸F]F0-PBB3 analog could be synthesized from a synthetic intermediateof a F0-PBB3 analog (see Table 2).

Synthesis Embodiment 59-2 Synthesis of1-[¹⁸F]fluoro-3-(2-((1E,3E)-4-(6-(methylamino)pyridine-3-yl)buta-1,3-dienyl)benz[d]thiazole-6-yloxy)propan-2-ol([¹⁸F]F0-PBB3)

[¹⁸F]F0-PBB3 can be synthesized from pre21.

Synthesis Embodiment 60 Synthesis of(E)-1-[¹⁸F]fluoro-3-(2-(4-(6-(methylamino)pyridine-3-yl)buta-1-en-3-ynyl)benz[d]thiazole-6-yloxy)propan-2-ol([¹⁸F]F0-PBB3.2)

[¹⁸F]F0-PBB3.2 can be synthesized from pre22.

Synthesis Embodiment 61 Synthesis of2-((1E,3E-4-(2-[¹⁸F]fluoro-6-(methylamino)pyridine-3-yl)buta-1,3-dienyl)benz[d]thiazole-6-ol([¹⁸F]F1-PBB3)

[¹⁸F]F0-PBB3.2 can be synthesized from pre23.

Synthesis Embodiment 62 Synthesis of(0-2-(4-(2-[¹⁸F]fluoro-6-(methylamino)pyridine-3-yl(buta-1-en-3-ynyl)benz[d]thiazole-6-ol([¹⁸F]F1-PBS3.2)

[¹⁸F]F1-PBB3.2 can be synthesized from pre24.

Synthesis Embodiment 63 Synthesis of2-((1E,3E)-4-(2-[¹⁸F]fluoro-6-(methylamino)pyridine-3-yl)buta-1,3-dienyl)benzofuran-5-ol([¹⁸F]F1-PBBf3)

[¹⁸F]F1-PBBf3 can be synthesized from pre25.

Synthesis Embodiment 64 Synthesis of(E)-2-(4-(2-[¹⁸F]fluoro-6-(methylamino)pyridine-3-yl(buta-1-en-3-ynyl(benzofuran-5-ol([¹⁸F]F1-PBBf3.2)

[¹⁸F]F1-PBBf3.2 can be synthesized from pre26.

Synthesis Embodiment 65 Synthesis of 24(1E,3E)-4-(6-(N-[¹¹C]methyl-N-methylamino)pyridine-3-yl(buta-1,3-dienyl)quinoline-6-ol([¹¹C]PBQ3.0)

[¹¹C]PBQ3.0 was synthesized by the same method as the methods shown insynthesis examples 42 and 43 above.

Synthesis Embodiment 66 Synthesis of24(1E,3E)-4-(6-([¹¹C]methylamino)pyridine-3-yl)buta-1,3-dienyl)quinoline-6-ol([¹¹C]PBQ3)

[¹¹C]H3Q3 was synthesized by the same method as the methods shown insynthesis examples 42 and 43 above.

Synthesis Embodiment 67 Synthesis of(E)-2-(4-(6-(N-[¹¹C]methyl-N-methylamino)pyridine-3-yl)buta-1-en-3-ynyl)quinoline-6-ol([¹¹C]PBQ3.1)

[¹¹C]PBQ3.1 was synthesized by the same method as the methods shown insynthesis examples 42 and 43 above.

Synthesis Embodiment 68 Synthesis of(E)-2-(4-(6-([¹¹C]methylamino)pyridine-3-yl(buta-1-en-3-ynyl)quinoline-6-ol([¹¹C]PBQ3.2)

[¹¹C]PBQ3.2 was synthesized by the same method as the methods shown insynthesis examples 42 and 43 above.

Biological Embodiments (Compounds and Reagents)

BSB and FSB were purchased from Doujindo. PIS and FDDNP were purchasedfrom ABX. Dimethylamino-styryl-benzothiazole and thioflavine-T werepurchased from Sigma-Aldrich. Thioflavine-S was purchased from Waldeck.BF-227, BF-158, THK523, and BF-189(N-methyl-4-[6-(quinoline-2-yl)hexa-1,3,5-trienyl]aniline) were providedfrom Tohoku University. Another β-sheet binding compound, whichcontained PBBS, BTA-1, BF-170, and curcumin, was purchased fromSigma-Aldrich. A potential amyloid ligand which contained cyanine,pyridine, pyridinium, benzothiazole, oxazine, thionine, and polyphenol,was purchased commercially. Dimethylsulfoxide (DMSO) was purchased fromSigma-Aldrich. Other chemical reagents were purchased commercially.

(Animal Models)

Human T34 (4-repeat tau isoform having one N-terminal insertion) heteroTg mice (also referred to as “PS19 mice”), which were driven by a mouseprion protein promoter (PrP) and which had a FTDP-17 P301S mutation wereprovided from the University of Pennsylvania. The PS19 mice werebackcrossed to a C57BL/6 background. Regarding the PS19 mice, referencemay be made to Yoshiyama, Y. et al. Synapse loss and microglialactivation precede tangles in a P301S tauopathy mouse model. Neuron 53,337-351 (2007). All the mice were managed and handled in accordance with“National Research Council's Guide for the Care and Use of LaboratoryAnimals” and the facility guidelines of the present inventors. Thisanimal experiment protocol has been authorized by the Animal EthicsCommittees of the National Institute of Radiological Sciences.

(Dissected Brain Tissues)

Postmortem human brains were obtained from autopsies performed on anAlzheimer's disease (AD) patient, a Pick's disease patient, aprogressive supranuclear palsy patient, a corticobasal degenerationpatient, and a frontotemporal lobar degeneration patient havingubiquitin-positive and tau-negative inclusions. Tissues were fixed in10% neutral buffered formalin, and embedded in paraffin blocks. Also,brains were sampled from the mice, and fixed in a phosphate buffersolution containing 4% paraformaldehyde. The tissue samples werecryo-preserved with a phosphate buffer solution containing 30% sucrose,and sliced inside a cryostat (HM560; Carl Zeiss).

Biological Embodiment 1 (In Vitro Fluorometric Binding Assay)

Aβ40 fibrils were obtained by incubating synthetic peptides (PeptideInstitute) at 37° C. for 72 hours. Recombinant T40 proteins werefiberized by incubating at 37° C. for 72 hours with 0.1 mg/ml ofheparin. Synthetic Aβ peptides (Peptide Institute) were dissolved inphosphate buffered physiological saline water (PBS; pH 7.4) such thatthe final concentration would become 100 μM, and the resultant solutionwas incubated at 37° C. for 72 hours. The resulting solution was dilutedto 50 μM, and an equivalent amount of compound (PBS containing 0 to 0.5mM of 1% DMSO) was added. After reacting at 37° C. for 1 hour, thesamples were evaluated using a microplate spectrometer (Safire; Tecan).Human T40 was expressed in Escherichia coli DE3, refined, and dialyzedagainst a 30 mM Tris-HCl buffer solution (pH 7.5). Recombinant tauproteins (1 mg/ml) that were separated by reverse-phase HPLC wereself-polymerized in a 30 mM Tris-HC 1 buffer solution containing heparin(0.1 mg/ml), at 37° C., for 72 hours. After that, the tau fibrils (1 μM)were reacted with an equivalent amount of compounds according to thepresent invention, and the resultant mixture was evaluated in the sameway as the analysis of binding to Aβ40. Regarding the fluorometric data,the binding saturation curve was created and the parameter estimationmethod was conducted using Prism software (GraphPad).

(Result)

The high affinity of PBB1 and PBB5 to tau pathologies was made clear bya fluorometric analysis using Aβ and tau filaments formed in a testtube.

TABLE 3 Table: Fluorescence and binding properties to synthetic A βpeptides and recombinant protein associations λ_(ex) & λ_(em) (nm) EC₅₀(nM) EC₅₀(Aβ)/ Compound Aβ40 T40 Aβ40 T40 EC₅₀(Tau) Thioflavin -T 445 &445 & 1,463 ± 459 818 ± 231 1.8 495 485 PBB5 635 & 630 & 1,217 ± 850 126± 67  9.7 685 685 PBB1 440 & 515 & 4,109 ± 764 402 ± 352 10.2 565 565

In this table, λ_(ex) and λ_(em) are respectively the optimal excitationwavelength and the detection wavelength in fluorescence microscopymeasurement of compounds that are bound to Aβ40 and T40 (the longest tauisoform formed with 441 amino acid residues) polymers. EC₅₀ (average±SE) is the effective concentration of the compounds, at which themaximum fluorescence intensity at the saturation point decreases byhalf. The ratio of the EC₅₀ of the Aβ40 fibrils to the EC₅₀ of the T40fibrils is shown in the rightmost column in the table.

Biological Embodiment 2 (In Vitro and Ex Vivo Fluorescence MicroscopyMeasurement, and Ex Vivo Multi-Photon Imaging)

6 μm paraffin sections from patients' brains, and 20 μm frozen sectionsfrom mouse brains were stained with 10⁻³% compounds (PIB, BF-158, FDDNP,BF-227, PBB1, PBB2, PBB3, PBB4, PBB5, curcumin, FSB, thioflavin-S, orBF-189) dissolved in 50% ethanol, at room temperature, for 1 hour.Images of fluorescence signals from these compounds were picked up usinga non-laser microscope (BZ-9000; Keyence Japan) and a confocal lasermicroscope (FV-1000; Olympus). In confocal imaging, theexcitation/emission wavelengths (nm) for each compound were optimized asfollows: 405/420-520 (PBB3, FSB, PIB, BF-227, BF-158, FDDNP,thioflavin-S), 488/520-580 (PBB2, PBB4), 515/530-630 (PBB1, curcumin),and 635/645-720 (PBBS, BF-189, DM-POTEB). Following this, test samplesand neighboring sections were processed by an autoclave for antigenactivation, immuno-stained with AT8 (Endogen) and an anti-Aβ N3 (pE)(pyroglutamylated Aβ3-x) polyclonal antibody, and analyzed using themicroscopes. For ex vivo imaging, PS19 mice and non-Tg WT mice, 10 to12-month old, were anesthetized with 1.5% (v/v) isoflurane, and 1 mg/kgof PBB1 to PBB4, 0.1 mg/kg of PBB5, or 10 mg/kg of FSB were administeredin the caudal vein. 60 minutes after the administration, the mice weredecapitated. Brain and spinal cord tissues were sampled, and cut intothin sections that were 10 μm thick, in a cryostat (HM560). The sectionswere imaged using the microscopes, labeled with FSB or AT8, and imageswere obtained again by the microscopes.

Ex vivo multi-photon imaging was performed as follows. The PS19 micewere given an intravenous injection of 1 mg/kg of PBB2 and PBB4,dissolved in 100 μl of physiological saline water containing 20% DMSO,and, 60 minutes after the administration, the brain and spinal cord wereextracted. After that, using a multi-photon laser light-receivingimaging system, a spinal cord sample was tested using 2-photonfluorescence that was generated from a pulse laser (Mai Tai;Spectra-Physics) in 800-nm excitation. The detection wavelength was made540 to 590 nm.

(Result)

FIGS. 1A-1B and FIG. 2 show fluorescence images of sections of an ADbrain having senile plaques and tau pathologies and a non-AD tauopathybrain characterized by tau aggregations but lacking senile plaques. Inthe AD brain, PBB1 to PBB5 strongly labeled NFTs, neuropil threads, andplaque neurites around senile plaques (FIG. 1), and furthermore stronglylabeled Pick bodies in Pick's disease, and tau aggregates in non-ADtauopathies such as neurological and glial fibrous lesions inprogressive supranuclear palsy (PSP) and corticobasal degeneration (CBD)(FIG. 2). On the other hand, the compounds other than PBB1 to PBB5provided insufficient labeling of these (FIGS. 1A-1B and FIG. 2). Notethat conventional amyloid stain thioflavin-S and FSB are known to havedifficulty passing the blood brain barrier (literature by Zhuang, Z. P.et al., Radioiodinated styrylbenzenes and thioflavins as probes foramyloid aggregates. J. Med. Chem. 44, 1905-1914 (2001).).

FIG. 3A shows in vitro and ex vivo labeling results of NFT-like tauinclusions in the PS19 mice using PBB1 to PBB5. Similar to thefluorescent labeling result of tau pathologies in the non-AD tauopathybrain, although the NFT-like inclusions in the brain stems and spinalcords of the PS19 mice were clearly identified with PBB1 to PBB5, thesewere not identified by other compounds that have been used in PETimaging heretofore (“in vitro” in FIG. 3A). In ex vivo labeling,although FSB was found to bind to tau accumulations in the PS19 mice(“in vivo” in FIG. 3A), a large amount of administration was necessaryfor this. To match these observation results, the 2-photon laserscanning fluorescence microscopic examination results of the ex vivosample showed that the spinal cord block of the PS19 mice was labelledwith PBB2 and PBB4 (the lowermost row in “in vivo” of FIG. 3A). Theseresults shown above indicate that the PBB compounds are sufficientlycapable of passing the blood brain barrier and cell membranes. As forthe other compounds, in vitro experiments, which were the same as theabove-described experiments performed on PBB1 to PBB5, were conducted,and the same results were achieved. These results are shown in FIG. 3B.

Biological Embodiment 3 (Non-Invasive Near Infrared Fluorescence Imagingof Tau Accumulations in Living Mouse Bodies)

(In Vivo and Ex Vivo Pulse Laser Scanning Imaging)

Non-invasive scanning of 12-month-old non-Tg WT mice and tau Tg mice,anesthetized with isoflurane, was performed using a smallanimal-dedicated optical imager (eXplore Optix; ART). Fluorescence wasgenerated from a 635-nm pulse laser diode (laser output, 25 to 125 mW,adjusted in each experiment; laser repetition rate, 80 MHz; pulse width,up to 100 ps) and detected with a 650-nm long pass filter and a fastresponse photomultiplier tube. In each experiment, the distance betweenthe top of the head and the detector was maintained constant by thehigh-precision vertical motion of the base and the side cameras. Themice were given an intravenous injection of 0.1 mg/kg of PBB5, dissolvedin 100 μl of physiological saline water containing 20% DMSO, and thehead parts of the mice were scanned, in a step width of 1.0 mm, and in aTPSF integration time of 0.1 to 0.3 seconds (optimized on a per scanbasis) per scan position. Dynamic imaging was performed over 240minutes, comprised of the baseline scan (before the administration), anda plurality of scans performed 5, 10, 15, 30, 45, 60, 90, 120, 180, 240,300, and 360 minutes after the injection. The fluorescence intensity wasstandardized between scans in accordance with the laser output and theintegration time. For each scan position, a TPSF curve was determined,and the time constant to match the exponential curve was estimated.Also, an ROI-based analysis was performed in parts of the headcorresponding to the frontal lobe, the brain stem, and the cervicalcord. The brains of these animals were extracted after in vivo assay andfixed with 4% paraformaldehyde, and 20 μm-thick frozen sections werestained with FSB and AT8.

(Result)

FIG. 4A shows a reference autofluorescent signal (center panel) laidover a visible light image (left panel) of the shaved head part ofnon-Tg WT mice. Elliptically-shaped regions of interest (ROIs) were setin the positions of the frontal cortex (FC), the brain stem (BS), andthe cervical cord (SC) (right panel). FIG. 4B shows fluorescenceintensity maps of PBB5 (0.1 mg/kg) in 12-month-old WT mice (upper part)and PS19 mice (lower part), before and 30 minutes and 240 minutes afterthe intravenous administration. The intensity maps were standardizedbased on the FC ROI values 30 minutes after the injection of PBB5. Nearinfrared fluorescence increased significantly immediately after the PBB5was administered, and, in 30 minutes, the fluorescence intensity in thebrain stem and spinal cord ROIs in the PS19 mice exceed the intensity inthe WT mice. Also, even 240 minutes later, PBB5 signals were observed inthe brain stems and spinal cords of the PS19 mice.

FIGS. 4C to 4E show the ratios of fluorescence intensity in the BS (c)and SC (d) ROIs, to the FC ROI, in the WT mice (white: n=7) and the PS19mice (black: n=7). These ratios were significantly bigger in the WT micethan in the PS19 mice (FIG. 4C and FIG. 4D: 2-way, repeated-measuresANOVA (time, F (11, 132)=17.6, p<0.001; region, F (1, 12)=29.9, p<0.001;genotype, F (1, 12)=23.6, p<0.001; FIG. 4E:*, p<0.05; **, p<0.01;Bonferroni's post hoc analysis). FIG. 4F shows a distribution diagram ofthe ratios of SC and BS to FC 240 minutes later, against the number ofFSB-positive NFT-like pathologies per unit area of 20-μm tissue sectionsof the tau Tg mice. The ratio of SC to FC in the PS19 mice 240 minuteslater showed a significant correlation with NFT-like tau pathologies inthe brain evaluated by FSB staining (FIG. 4F). This formed the basis ofthe applicability of this ratio to optical measurement as an in vivoindicator of tau accumulations.

FIG. 4G shows the fluorescence intensity (left) and the fluorescenceduration (right) in 11-month-old WT mice (upper part) and PS19 mice(lower part) 120 minutes after the intravenous injection of PPB5. The BSand SC ROIs of the tau Tg mice showed extended durations of fluorescencecompared to the WT mice (see the arrows). In the FC ROIs of the WT andTg mice, the fluorescence intensity increased remarkably, but thefluorescence duration thereof did not change much. FIG. 4H shows TPSFcurve of SC and FC spots 120 minutes after injection in 11 month-old WTmice and Tg mice. Compared to the WT data, an obvious delay offluorescence decay was observed in Tg SC.

FIG. 4I shows average durations of fluorescence (*: p<0.05; 2-wayrepeated-measures ANOVA with Bonferroni's post hoc analysis) in the FC,BS, and SC ROIs in the WT mice (white; n=7) and Tg mice (black; n=7) 120minutes after the injection. FIG. 4J shows a distribution diagram of thefluorescence duration periods in the BS and SC ROIs 120 minutes afterthe injection, against the number of FSB-positive NFT-like pathologiesper unit area in 20 μm-thick tissue sections of the Tg mice. The averagefluorescence duration periods in the brain stems and the spinal cords ofthe PS19 mice increased significantly compared to those of the non-Tg WTmice, and had a significant correlation with the number of NFTpathologies in the BS and SC ROIs. A TPSF curve can be considered to beformed with signals from compounds that are not bound or are boundnon-specifically, and that have short fluorescence duration, and taupathology-binding compounds that have extended fluorescence durationdepending on the growth of fibrils, so that the time constant that isdetermined by fitting this curve to the exponential function iseffective as a reasonable reliable indicator of the amount ofaccumulation of tau aggregates.

Biological Embodiment 4 (In Vivo 2-Photon Laser Scanning FluorescenceMicroscopic Examination)

12-month-old WT mice and PS19 mice were anesthetized with 1.5% (v/v)isoflurane, and their thoracic vertebrae were laminectomized. Coverglass was placed over spinal cord tissues, and the vertebral columnswere fixed with a Narishige STS-A spinal cord clamp and a MA-6Nhead-fixing adaptor. 12 mg/kg of sulforhodamine 101 (MP Biomedicals) wasadministered intraperitoneally, and, 15 minutes later, 1 mg/kg of PBB3was administered intravenously, and biological 2-photon fluorescenceimaging was performed. The detection wavelengths for PBB3 andsulforhodamine 101 were made 500 to 550 nm and 573 to 648 nm,respectively.

(Result)

FIGS. 5A through 5I show real-time 2-photon laser scanning images.Within 3 seconds after the injection of PBB3, PBB3 signals appeared inblood vessels that had been labeled in advance with sulforhodamine 101,and, in the next 5 minutes, the signals spread from the blood vessels tospinal cord tissues (FIG. 5A to FIG. 5F). After that, although PBB3 thatwas not bound was discharged from the spinal cord tissues, at the sametime, clear binding to tau inclusions (FIG. 5G and FIG. 5H, cuneiformsymbols) was shown. On the other hand, in the WT mice, such signals tooriginate from binding compounds were not observed. This resultindicates that PBB3 passes the blood brain barrier and quickly labelsthe tau deposits in the brain.

Biological Embodiment 5 (Autoradiography and PET Imaging of TauPathologies in PS19 Mice by Radio-Labeled Compounds)

(In Vitro Autoradiography)

12 to 15-month-old non-Tg WT mice and PS19 mice were decapitated, andtheir brains were frozen and sliced into 20 μm-thick sections in acryostat (HM560). The sections were placed on slide glass (MatsunamiGlass), and kept at −80° C., until an analysis. Similarly, sections ofthe cerebral cortex were obtained from an AD patient. Tissue sectionswere incubated for at room temperature for 60 minutes, in a 250 mMTris-HCl buffer solution (pH 7.4), containing 20% ethanol and [¹¹C]PBB2,or 10% ethanol and [¹¹C]PBB3 (37 MBq/L, up to 1 nM). Non-specificbonding was detected in the presence of a 10 μMV non-radioactive ligand.Samples were reacted with [¹¹C]PBB2 or [¹¹C]PBB3, and were each washedtwice, for 2 minutes, with an ice-cool Tris-HCl buffer solutioncontaining 20% or 10% ethanol, and immersed in ice water for 10 seconds.After that, the sections were dried with warm air, and placed on imagingplates (Fuji Film). The imaging plates were scanned by a BAS500 system(Fuji Film), and autoradiograms were obtained (FIG. 6A).

(Ex Vivo Autoradiography)

Under anesthesia with a 1 to 1.5% (v/v) isoflurane mixture (flow rate 2mL/minute), [¹¹C]PBB2 or [¹¹C]PBB3 (up to 37 MBq) was injected in thecaudal veins of 12 to 15 month-old non-Tg WT mice and PS19 mice. 45minutes after the injection, the mice were decapitated, and their brainswere quickly extracted and frozen with powder dry ice. The frozen braintissues were cut into 20 μm-thick sections with a cryotome. After that,autoradiograms were obtained (FIG. 6B). Also, the brain sections of thePS19 mice after autoradiography were stained with FBS.

(In Vivo PET (Positron Emission Tomography) Imaging of Mice)

PET scanning was performed using a micro PET focus 220 animal scanner(Siemens Medical Solutions), which provided 95 slices that were 0.851mm-thick (between centers), a 19.0-cm axial field of view (FOV) and a7.6-cm cross-sectional FOV. Before scanning, 9 to 15-month-old PS19 miceand non-Tg WT mice were anesthetized with 1.5% (v/v) isoflurane. Anemission scan was performed immediately after the intravenous injectionof [¹¹C]PBB2 (28.3±10.3 MBq), [¹¹C]pBB3 (29.7±9.3 MBq), or [¹¹C]mPBB5(32.8±5.9 MBq), for 90 minutes, in 3D list mode, with an energy window350-750 keV. The injection of the radioactive compound and scanning wereconducted under dim light so as to avoid photoracemization of thecompound. The entire list mode data was sorted into 3D sinograms, and,after that, converted into 2D sinograms by Fourier-rebining (frame:10×1, 6×5, and 5×10 minutes). Arithmetic mean images from 30 to 60minutes and 60 to 90 minutes after the injection of the radioactivecompound were obtained by maximum a posteriori reconstruction. Also,dynamic images were reconstructed by filtered back projection, using a0.5-mm Hanning filter. The volume of interest (VOI) was set in aplurality of anatomical structures, including the brain stem and thestriatum, using PMOD image analysis software (PMOD Technologies), withreference to an MRI template. With a subgroup of 12 month-old PS19 Tgmice that were subjected to [¹¹C]PBB3-PET scanning, TSPO dynamic PETimaging was performed over 90 minutes after an intravenous injection of[¹¹C]Ac5216 (34.6±8.8 MBq). [¹¹C]Ac5216-PET scanning was performedwithin one week after the [¹¹C]PBB3-PET scanning (FIG. 6C).

(Result)

FIG. 6A shows in vitro autoradiograms of the cerebellar brain stem partsand the AD frontal cortexes of the PS19 and non-Tg WT mice. With[¹¹C]PBB2 and [¹¹C]PBB3, fibrous aggregate pathologies in the brainstems and AD grey matters of the mice were strongly radio-labeled. Also,binding of [¹¹C]PBB3 was blocked by addition of non-radioactive PPB3 (10μM). FIG. 6B shows ex vivo autoradiogram of the PS19 and non-Tg WT mice,and FBS stain image diagrams of the PS19 brain slice. The arrowsindicate the brain stems containing many tau inclusions. With [¹¹C]PBB2and [¹¹C]PBB3, tau inclusions contained in the brain stem and spinalcord of the PS19 mice were radio-labeled. [¹¹C]PBB3 radio-labeled tauinclusions more selectively.

FIG. 6C shows sagittal-plane and coronal-plane PET images and MRIimages, obtained by averaging the dynamic scan data from 60 to 90minutes after the intravenous administration of [¹¹C]PBB3. The arrowsand the asterisks show the brain stem and the striatum, respectively,and the cuneiform symbol shows strong radiolabeling in the inner brainstem of the PS19 mice. FIGS. 6A and 6B show sagittal section PET imagesobtained by averaging dynamic scan data 60 to 90 minutes after theadministration of [¹¹C]PBB2. Tau pathologies of the PS19 mice weresuccessfully visualized in vivo.

FIG. 6D shows FSB stain images of a brain section extracted from thePS19 mice after PET scanning (a sagittal plane image (left panel) and acoronal plane image (center panel), and a high-magnification image(right panel)) of fibrous tau inclusions. It is shown that thetopographies of PET signals and NFT-like tau inclusions match in thePS19 mice.

FIG. 6E shows the time-activity curves (left panel) in the striatums(ST) and brain stem (BS) of the PS19 mice and WT mice, and, the BS-to-STratios of radioactivity (right panel) (in each, n=5). After theintravenous injection, [¹¹C]PBB3 passed the blood brain barrier quickly,and [¹¹C]PBB3 that was not bound and that was non-specifically bound wasimmediately removed from the brains at a half-life of approximately 10minutes. Also, the [¹¹C]PBB3 signal in the brain stems of 12-month-oldPS19 mice was maintained over the imaging period (90 minutes), and thiswas significantly different from the result of non-Tg WT mice of thesame month age (FIG. 6E, the left panel). The striatum (ST) lacking taupathologies was used as a reference region, and the ratio of the targetbrain stem (BS) to that reference region marked the maximum value inapproximately 70 minutes (right panel, FIG. 6E). On the other hand, inthe WT mice, this kept decreasing over 60 minutes. Compared to12-month-old WT mice, the average ratio over 45 to 90 minutes increasedby 40% in PS19 mice of the same month age.

FIGS. 6H and 6I show ex vivo autoradiography images of the mice shown inFIGS. 6F and 6G. The arrows in the drawings show an increase ofradiolabeling in PS19 mice. FIGS. 6J and 6K show FSB stain images, usingthe same samples as the samples from which the autoradiography imagesare obtained. FIG. 6L shows time-activity curves in a plurality of braintissues of WT mice. FIG. 6M shows the ratios of radioactivity in thebrain stem to the striatum, in PS19 mice (1 in the drawing) and WT mice(2 in the drawing) (n=5), over the imaging period.

FIG. 7 shows coronal-plane PET images in the brains of the WT mice (leftpanel) and the PS19 Tg mice (right panel) given an injection of[¹¹C]mPBB5. These images were laid over an MRI template by averaging thedynamic data from 30 to 90 minutes after the injection. The PET imagesshow that a lot of [¹¹C]mPBB5 was held in the brain stems of the PSmice, compared to the WT mice.

Biological Embodiment 6 (In Vitro Autoradiography of AD Brains IncludingHuman Hippocampal Formations)

In order to compare the binding of [¹¹C]PBB3 and [¹¹C]PIB to areasinside the human brain where there were plenty of tau pathologies, invitro autoradiograms were obtained using AD brain slices including thehippocampal formation.

(In Vivo PET Imaging of Humans)

2 subjects with normal cognitive function (72 years old and 75 yearsold; average 73.5 years old), and 3 AD patients (64 years old, 75 yearsold and 77 years old; average 72 years old) were employed for thisstudy. All the subjects were males, and all the AD patients werediagnosed in accordance with the standards of the National Institute ofNeurological and Communicative Diseases and Stroke/Alzheimer's Diseaseand Related Disorders Association (NINCDS-ADRDA). The clinical dementiarating scale was 0 for both normal subjects, and ranged from 1 to 2 withthe AD patients. Their cognitive function was evaluated by a mini-mentalstate examination (MMSE). No subject showed MRI-based brainabnormalities. On the other hand, the AD patients exhibited atrophy ofthe neocortex and the hippocampus. This clinical study had beenauthorized by the Ethics and Radiation Safety Standards Committee of theNational Institute of Radiological Sciences. Informed consent had beenobtained from the subjects or from their family. PET assay was performedusing a Siemens ECAT EXACT HR+ scanner (CTI PET Systems) with an axialFOV of 155 mm, 63 consecutive 2.46 mm-thick slices, and an axialresolution of 5.4 mm with a tangential resolution of 5.6 mm. In order tomeasure tissue attenuation, a transmission scan was performed for 10minutes, and dynamic emission scan data was collected in 3D mode, over70 minutes immediately after an intravenous injection of [¹¹C]PIB(350±50 MBq). A plurality of image frames (3×20, and 3×40 seconds, and1x 1, 2×3, 5×6, and 3×10 minutes) were obtained from that dynamic scan.Similarly, with the same individuals, a second PET session using[¹¹C]PBB3 was performed approximately 2.5 hours after [¹¹C]PIB-PET wasfinished. [¹¹C]PBB3 (370±50 MBq) was injected in the vein over 60seconds, and emission data was obtained in 70 minutes (frames: 3×20, and3×40 seconds, and 1×1, 2×3, 5×6, and 3×10 minutes). During the[¹¹C]PBB-PET scanning, artery blood samples are obtained 10, 20, 30, 40,50, 60, 70, 80, 90, 100, and 110 seconds after the injection, and 2,2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, 60 and 70minutes after the injection, and the amount of radioactivity in theplasma was measured. The radioactivity to match [¹¹C]PBB3 inunmetabolized plasma was measured by HPLC using the samples from 3, 10,20, 30 and 60 minutes after the injection (Waters mBondapak C18 column,7.8 mm×300 mm; acetonitrile/ammonium formate mobile phase, gradientelution=40/60 (0 minutes), 52/48 (6 minutes), 80/20 (7 minutes), 80/20(8 minutes), 40/60 (9 minutes) and 40/60 (15 minutes); flow rate, 6mL/minute). The injection of the radioactive compound and the followingscan, and the plasma assay were performed under dim light so as to avoidphotoracemization of the compound.

Individual MRI data was recorded simultaneously with PET images, usingPMOD software package (PMOD Technologies). VOIs were set in the MRimages recorded simultaneously, and moved to the PET images. The VOIswere defined in the cerebellar cortex, the middle temporal areaincluding the parahippocampal gyrus and the hippocampus, the basal partside of the frontal cortex, the precuneus part of the parietal cortex,and the centrum semiovale. Each VOI included three neighboring slices,and, by combining the data, an average radioactivity concentration ofall VOIs was obtained. A standardized uptake value (SUV) was calculatedfrom the time-integrated regional radioactivity concentrationstandardized by the injection dose/weight. The integration interval wasset on the data of 30 to 70 minutes. The cerebellum could be used as areference brain region, so that the SUV ratio (SUVR) of the cerebellumwas measured for each target VOI, as an indicator of senile plaques ortau depositions.

(Result)

FIG. 8A shows autoradiography of an AD patient's brain slice using 10 nMof [¹¹C]PBB3 (left) and [¹¹C]PIB (center). This section includes thehippocampus (Hi), the parahippocampal gyrus (PH), the fusiform gyrus(FF), and the white matter (asterisk). Total binding of [¹¹C]PBB3 and[¹¹C]PIB was clearly discarded, except for the white matter that waslabeled with [¹¹C]PIB, by addition of non-radioactive PBB5 (100 μM) andthioflavin-S (10 μM) (NS). Strong [¹¹C]PBB3 signals were observed in thehippocampus CAI region and the pes hippocampi, but no [¹¹C]PIB signalwas observed. Also, there was more binding of [¹¹C]PBB3 in the cortexregion (black dot) in the side of the collateral sulcus, compared to thebinding of [¹¹C]PIB. The FSB stain of amyloid fibrils in this sectionshowed that there were many NFT pathologies in the CAI and the subiculum(Sub), and that there were many senile plaques in the fusiform gyrus(FF) (right panels). This suggests strong reactivity of [¹¹C]PBB3 toNFTs in AD brains.

FIG. 8B shows MRI images (left) and PET images using [¹¹C]PBB3 (center)and [¹¹C]PIB (right), taken from the same AD (upper part) and normalcontrol (NC; lower part) subjects. The coronal section images includethe hippocampal formation (cuneiform symbols). Compared to the NC,although the [¹¹C]PBB3 signal increased in the hippocampal formation ofthe AD patient, the [¹¹C]PIB signal did not change much. This indicatedthat, unlike [¹¹C]PIB, [¹¹C]PBB3 bound strongly with NFTs in the ADpatient's hippocampus.

Abbreviations

AD: Alzheimer's diseaseAIBN: azobisisobutyronitrileAT8: anti-phospho-tau antibodyBF-158: 2-[(4-methylamino)phenyl]quinolineBF-170: 2-(4-aminophenyl)quinolineBF-189: N-methyl-4-[6-(quinoline-2-yl)hexa-1,3,5-trienyl]anilineBF-227:2-(2-[2-dimethylaminothiazole-5-yl]ethenyl)-6-(2-[fluoro]ethoxy)benzoxazole)BSB: (E,E)-1-bromo-2,5-bis(3-hydroxycarbonyl-4-hydroxy)styrylbenzeneBTA-1: 2-(4-methylaminophenyl)benzothiazole

DM-POTEB:

2-[8-(4-dimethylaminophenyl)octa-1,3,5,7-tetraenyl]-3-ethylbenzothiazole-3-iumFDDNP: 2-(1-{6-[(2-fluoroethyl)(methyl)aminol-2-naphthyl}ethylidyne)malononitrileFSB: (E,E)-1-fluoro-2,5-bis(3-hydroxycarbonyl-4-hydroxy)styrylbenzeneFTDP-17: frontotemporal dementia linked to chromosome 17 withParkinsonismMRI: magnetic resonance imagingNFT: neurofibrillary tangle

NBS: N-bromosuccinimide

PET: positron emission tomography

PIB: Pittsburgh Compound B

T40: the longest tau isoform formed with 441 amino acid residuesTBDMSCl: tert-butyldimethylchlorosilaneTg: transgenicTHK523: 2-(4-aminophenyl)-6-(2-fluoroethoxy)quinolineTSPO: translocator proteinWT: wild type

INDUSTRIAL APPLICABILITY

The compounds of the present invention can be used to clarify themechanism by which tau proteins to accumulate in the brains of patientsof diseases such as Alzheimer's disease, frontotemporal lobardegeneration, dementia, and other neurodegenerative tauopathies areproduced. Also, by using the compounds of the present invention, it ispossible to diagnose the above diseases, predict future episodes, andperform screening of candidate compounds for treatment of the abovediseases. Furthermore, by using the compounds of the present invention,it is possible to plan strategies for treatment of the above diseases.

1. A compound of Formula (I), a pharmaceutically acceptable saltthereof, or a solvate thereof:

wherein: R₁ and R₂ are each independently selected from the groupconsisting of hydrogen, alkyl, alkenyl, acyl, and hydroxyalkyl; R₃ ishydrogen or halogen; ring A is a benzene ring or a pyridine ring; ring Bis selected from the group consisting of the following formulas (i),(ii), (iii), and (iv):

in the formula (ii), R_(a) is alkyl; R₄ and R₅ are each separatelyselected from the group consisting of hydrogen, hydroxy, alkoxy,haloalkoxy, halohydroxyalkoxy, and aminoalkyl, wherein R₄ and R₅ are notsimultaneously hydrogen if ring B is formula (i) or (ii); and

represents a double bond or a triple bond.
 2. The compound according toclaim 1, wherein ring B is formula (i) or (ii).
 3. The compoundaccording to claim 1, wherein ring A is a pyridine ring.
 4. The compoundaccording to claim 3, represented by the following formula (II):

wherein R₁ to R₅ and

are defined in claim
 1. 5. The compound according to claim 4, wherein

represents a double bond.
 6. The compound according to claim 4, wherein

represents a triple bond.
 7. The compound according to claim 1, whereinring A is a benzene ring.
 8. The compound according to claim 7,represented by the following formula (III):

wherein R₁ to R₅ and

are defined in claim
 1. 9. The compound according to claim 8, wherein

represents a double bond.
 10. The compound according to claim 8, wherein

represents a triple bond.
 11. The compound according to claim 7,represented by the following formula (IV):

wherein R₁ to R₅, R_(a) and

are defined in claim
 1. 12. The compound according to claim 11, wherein

represents a double bond.
 13. The compound according to claim 11,wherein

represents a triple bond.
 14. The compound according to claim 1, whereinring B is formula (iii).
 15. The compound according to claim 14,represented by the following formula (V):

wherein R₁ to R₅ and

are defined in claim
 1. 16. The compound according to claim 15, wherein

represents a double bond.
 17. The compound according to claim 15,wherein

represents a triple bond.
 18. The compound according to claim 1, whereinring B is formula (iv).
 19. The compound according to claim 14,represented by the following formula (VI):

wherein R₁ to R₅ and

are defined in claim
 1. 20. The compound according to claim 19, wherein

represents a double bond.
 21. The compound according to claim 19,wherein

represents a triple bond.
 22. A compound, a pharmaceutically acceptablesalt thereof, or a solvate thereof, selected from the following group:


23. The compound according to claim 1, wherein one or more atoms are aradioisotope of the atom or atoms.
 24. A composition comprising acompound according to claim 1, and a pharmaceutically acceptablecarrier.
 25. A composition for tau imaging, comprising a compound ofrepresented formula (I),

wherein: R₁ and R₂ are each separately selected from the groupconsisting of hydrogen, alkyl, alkenyl, acyl, and hydroxyalkyl; R₃ ishydrogen or halogen; ring A is a benzene ring or a pyridine ring; ring Bis selected from the group consisting of the following formulas (i),(ii), (iii), and (iv):

in the formula (ii), R_(a) is alkyl; R₄ and R₅ are each separatelyselected from the group consisting of hydrogen, hydroxy, alkoxy,haloalkoxy, halohydroxyalkoxy, and aminoalkyl; and

represents a double bond or a triple bond; or a pharmaceuticallyacceptable salt thereof, or a solvate thereof; and a pharmaceuticallyacceptable carrier.
 26. The composition for tau imaging according toclaim 25, wherein, in the compound, one or more atoms are a radioisotopeof the atom or atoms.
 27. An intermediate for synthesizing the compoundaccording to the formula (I), represented by one of the followingformula (I-i), (I-ii), (I-i) protector 1, (I-i) protector 2, (a6)protector, (a7), (a8-1), or (a8-2):

wherein R₃, R₄, R₅ and

are defined in claim
 1. 28. The intermediate of claim 27 forsynthesizing the compound according to the formula (I), represented bythe following formula (I-ii):

wherein R₁, R₂, R₃, R₄, R₅, and

are defined in claim
 1. 29. The intermediate of claim 27 forsynthesizing the compound according to the formula (I), represented bythe following formula (I-i) protector 1:

wherein R₃, R₅ and

are defined in claim
 1. 30. The intermediate of claim 27 forsynthesizing the compound according to the formula (I), represented bythe following formula (I-i) protector 2:

wherein R₃, R₅,

and are defined in claim
 1. 31. The intermediate of claim 27 forsynthesizing the compound according to the formula (I), represented bythe following formula (a6) protector:

wherein R₁, R₅, and

are defined in claim
 1. 32. The intermediate of claim 27 forsynthesizing the compound according to the formula (I), represented bythe following formula (a7):

wherein R₁ to R₃, R₅, and

are defined in claim
 1. 33. The intermediate of claim 27 forsynthesizing the compound according to the formula (I), represented bythe following formula (a8-1) or (a8-2):

wherein R₁ to R₃, R₅, and

are defined in claim 1, and Alk is alkyl.
 34. The intermediate of claim27 for synthesizing the compound according to claim 1, selected from thefollowing group: